Inﬂuence of Metal-Resistant Staphylococcus aureus Strain K1 on the Alleviation of Chromium Stress in Wheat

: Chromium (Cr) is recognized as a toxic metal that has detrimental e ﬀ ects on living organisms; notably, it is discharged into soil by various industries as a result of anthropogenic activities. Microbe-assisted phytoremediation is one of the most emergent and environmentally friendly methods used for the detoxiﬁcation of pollutants. In this study, the alleviative role of Staphylococcus aureus strain K1 was evaluated in wheat ( Triticum aestivum L.) under Cr stress. For this, various Cr concentrations (0, 25, 50 and 100 mg · kg − 1 ) with and without peat-moss-based bacterial inoculum were applied in the soil. Results depicted that Cr stress reduced the plants’ growth by causing oxidative stress in the absence of S. aureus K1 inoculation. However, the application of S. aureus K1 regulated the plants’ growth and antioxidant enzymatic activities by reducing oxidative stress and Cr toxicity through conversion of Cr 6 + to Cr 3 + . The Cr 6 + uptake by wheat was signiﬁcantly reduced in the S. aureus K1 inoculated plants. It can be concluded that the application of S. aureus K1 could be an e ﬀ ective approach to alleviate the Cr toxicity in wheat and probably in other cereals grown under Cr stress. Project administration, F.Z., T.Y. and S.A.; Resources, F.Z., M.W., S.A., M.A.E.-S., M.N.A. and L.W.; Software, F.Z., M.Z., M.A.E.-S. and L.W.; Supervision, M.R., T.Y. and S.A.; Validation, M.Z., M.W., A.A. (Alia Anayat) and A.A. (Awais Ahmad); Visualization, M.Z., M.W., A.A. (Alia Anayat) and A.A. (Awais Ahmad); Writing—original draft, F.Z., M.Z., S.A. and L.W.; Writing—review & editing, F.Z., T.Y. and M.N.A.


Introduction
Environmental pollution by toxic metals has dramatically increased because of various man-made actions taken while revolutionizing industries and urban life. Although these activities have substantially improved the living standards of humans, they have, at same time, deteriorated the environment [1]. Direct or indirect discharge of sewage and industrial effluent into surface water bodies has resulted in augmentation of chromium (Cr) and other toxic metals in soils [2], causing toxicity to

Soil Preparation
Sandy clay loam soil was brought from nursery and was air-dried without direct sunlight. After air-drying, soil sieving was done by a mesh with a pore size of 2 mm. Soil was then sterilized at a temperature of 121 • C for 20-30 min for the purpose of removing any kind of contaminant or bacteria that can cause hindrance in further findings [31]. Chromium solutions of different concentrations were prepared from stock solution of K 2 Cr 2 O 7 in the laboratory, and soil was spiked with final Cr concentrations of 0, 25, 50 and 100 mg·kg −1 of soil.
These different concentrations of Cr were taken to determine the maximum concentration of hexavalent Cr tolerable by strain K1. However, in case of Cr reduction, the lower concentration of Cr was used due to the fact that Cr is found in lower concentrations in the natural environment, especially in industrial effluents [31]. The concentrations of Cr used were similar to those used in the literature and were chosen considering the fact that, in field conditions, we had to establish the reduction ability of this particular strain rather than its maximum potential to survive in response to metal stress [15]. The soil was added in the pots (5 kg soil per pot) with proper mixing following the treatment plan. Electrical conductivity and pH from saturated soil were determined by making a soil-to-water ratio of 1:25. Soil was extracted with ammonium bicarbonate diethylenetriaminepentaacetic acid (AB-DTPA) solution for the measurement of bioavailable trace elements in the soil [32]. Soil organic matter was determined following the prescribed method [33]. Soil physicochemical characteristics are given in Table 1.

Segregation of Cr-Resistant Bacteria
A modified method of serial dilution was adopted to isolate the Cr-tolerant bacteria from metal-contaminated industrial effluent [34]. For this, ten-fold serial dilutions (10 −1 , 10 −2 , 10 −3 and 10 −4 ) were prepared from samples of collected wastewater using sterilized distilled water [34]. Then, 0.1 mL from each dilution was added to petri plates having Tryptic Soy Agar complemented with 0.5 mM Cr 6+ . Morphologically different colonies were picked and transferred to petri plates supplemented with gradually elevated levels (0.0, 0.5, 2.5, 5.0, 10.0, 15.0, 20, 22 and 23 mM) of Cr 6+ [35]. The bacteria Agronomy 2020, 10, 1354 4 of 18 that showed maximum resistance to the highest concentration of hexavalent Cr were selected for use in further studies.

Bacterial Identification
Molecular characterization was carried out through the amplification of 16S rDNA gene via polymerase chain reaction (PCR) using the following universal primers: 27F (5 -AAACTCAAATGAATTGACGG-3 ) and 1492R (5 -ACGGGCGGTGTGTAC-3 ) [36]. For genomic DNA extraction, Favorgen DNA extraction kit was used following the manufacturer's guideline. The initial denaturation temperature was set at 94 • C for a period of 5 min, and this was followed by 40 recurring cycles of denaturizing DNA at 94 • C for 45 s, annealing at 53 • C for 45 s and elongation at 72 • C for 60 s. Final extension was set at 72 • C for 10 min, and this was followed by temperature being held at 4 • C [37]. PCR product (5 µL) was loaded in gel wells, and the reaction was allowed to complete; the product was then visualized using Gel Documentation System (Slite 200 W) under ultraviolet light [37]. After validation, 30 µL PCR product was delivered to Macrogen (Seoul, Korea) for the purpose of sequencing. ChormasPro (v1.7.1) software was used for correction of sequences that were submitted to GenBank for accession number. A phylogenetic tree was constructed by downloading similar partial 16S rDNA gene sequences from the NCBI BLAST database with the help of computer software MEGA (v7.0.) [38].

Bacterial Inoculum Preparation
In order to obtain pure inoculum of S. aureus strain K1, an individual isolated colony was inoculated in 250 mL sterilized nutrient broth and incubated at 150 rpm on orbital rotary shaker for 48 h (at 37 • C). The pure culture was harvested via centrifugation at 6000× g for 10 min, and the supernatant was discarded. The pellet was washed with sterilized distilled water and resuspended in 100 mL of normal saline (0.85% NaCl) solution. Overall, cell density for the inoculum was maintained at 1 × 10 8 CFU mL −1 [39].

Seed Coating and Pot Experiment
For this study, seeds of wheat variety Sehar were taken from Ayub Agriculture Research Institute, Faisalabad, Pakistan. Seeds were first washed thoroughly with distilled water, and this was followed by surface sterilization using 10% hydrogen peroxide (H 2 O 2 ) for 30 min [40]. The sterilized seeds were immersed in double volume of bacterial suspension (1 × 10 8 CFU mL −1 ) and kept at 37 ± 2 • C on a rotary shaker (90 rpm) for 2 h. To facilitate the attachment of bacterial inoculum to the seeds, carboxymethyl cellulose (CMC) (2%) was added to the suspension as a sticking agent. Seeds were dried under shade after 2 h of inoculation for further experimental use. Uninoculated sterilized seeds were used as control. Clay and peat moss in equal parts (1:1) were mixed and the seeds were added to this mixture, which was shaken well for proper coating and incubated overnight in the dark. The completely randomized design had a total of eight treatments, with three replicates for each treatment. A total of eight seeds per pot were sown, and thinning was performed to result in four seedlings per pot after 3 weeks of seed germination.

Plant Harvesting
At 135 days after seed sowing, plants were harvested at maturity. The height and spike lengths of plants were measured with a meter rod. Shoots, roots, spikes and grains were separated properly. Then, 0.1 M HCl was used to remove the metals from the root surface, and the roots were washed with distilled water. Samples of roots and shoots were kept in a hot air oven (70 • C) for a period of 72 h. Afterwards, dry weight was recorded and samples were crushed to small pieces and processed for further analyses.

Determination of Chlorophyll Contents and Gas Exchange Parameters
At 8 weeks after seed germination, fresh leaf samples were taken to determine chlorophyll contents using acetone (85% v/v) for pigment extraction. These leaf samples were kept in the dark at 4 • C for 24 h. Centrifugation of samples was done to get the supernatant. Absorbance was recorded by spectrophotometer at three different wavelengths (470, 647 and 664.5 nm), and final chlorophyll contents were calculated by following the prescribed method [41]. Photosynthetic rate, transpiration rate and stomatal conductance of samples were recorded 8 weeks after seed germination on a fully sunny day using an infrared gas analyzer (IRGA, LCA-4, Analytical Development Company, Hoddesdon, UK).

Determination of Reactive Oxygen Species and Antioxidant Enzyme Activities
At 2 months after seed sowing, fresh leaves of plants were sampled for the estimation of reactive oxygen species (ROS) through the assessment of electrolyte leakage (EL) and the contents of malondialdehyde (MDA) and hydrogen peroxide (H 2 O 2 ). Additionally, the activities of enzymes such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) were assessed. For the EL estimation, distilled water tubes were used to place leaf samples. Samples were autoclaved at 32 • C for period of 2 h, and the observed EC of the solution was termed as EC 1 . Afterwards, this solution was autoclaved at 121 • C for 20 min to measure EC 2 , and finally EL was calculated using the following equation as described by Dionisio-Sese and Tobita [42]: The concentration of MDA was measured using the method of Heath and Packer (1968) as modified by Dhindsa et al. [43] and Zhang and Kirham [44]. Hydrogen peroxide was recorded through homogenization of samples in phosphate buffer 50 mM (pH 6.5) and centrifugation followed by addition of 20% H 2 SO 4 (v/v). Samples were centrifuged once more for 15 min, and readings were taken by spectrophotometer at 410 nm absorbance [45]. A spectrophotometer was utilized to record the activities of antioxidant enzymes such as SOD, POD, CAT and APX. Fresh leaf samples were crushed in liquid nitrogen (N 2 ), and 0.05 M phosphate buffer (pH 7.8) was utilized for the purpose of standardization. This was followed by centrifugation at 4 • C on 12,000× g for a period of 10 min. Supernatant was collected for the sake of antioxidant enzyme activity measurements. The method of Zhang [46] was employed to measure SOD and POD activities, while the Aebi method [47] was used for CAT activity. APX contents were estimated using the method of Nakano and Asada [48].

Estimation of Cr Contents in Plants
Digestion of dry shoot and root samples was performed for 1 g of each sample in 4:1 (v/v) ratio of HNO 3 :HClO 4 as described by Rehman et al. [49]. Finally, the digested samples were run on an atomic absorption spectrophotometer for the estimation of Cr concentrations in the processed samples.

Statistical Analysis
The IBM SPSS Statistics for Windows, Version 21.0, was used for the data analyses, using the analysis of variance (ANOVA) tool at a 5% probability level. Tukey's HSD post hoc test was performed for multiple comparison of triplicates.

Results
The current study was envisaged to assess the capability of metal-resistant Staphylococcus aureus strain K1 to ameliorate the Cr stress in wheat plants.

Growth Characteristics of Isolate K1
The bacterial strain K1, capable of tolerating a Cr concentration of up to 22 mM, was selected for further studies. Morphologically, it is characterized by Gram-positive cocci (≈1 µm) with yellowish golden color. Chemically, it is oxidase-and coagulase-negative and catalase-positive ( Table 2). The BLASTn investigation showed that it has a close resemblance (99%) to Staphylococcus aureus strain ATCC 12600 (NR_115606.1) and Staphylococcus aureus strain NBRC 100910 (MG971399.1). The similar 16S rDNA gene sequences from GenBank were used to carry out phylogenetic analysis, which also confirmed that the isolate K1 belongs to Staphylococcus aureus; therefore, it was named Staphylococcus aureus strain K1 (KX685332). This was done in order to remain confident that the bacterial strain used in this study was Staphylococcus aureus strain K1, as culture media can sometimes be contaminated with other bacteria. Staphylococcus aureus K1 exhibited optimum growth at pH 8 and 35 • C. Under optimum growth conditions, the effect of contact time on bacterial ability to reduce the hexavalent Cr in the medium was observed. It was observed that the Cr reduction of S. aureus K1 increased with increasing contact time ( Figure 1). It was found that 26%, 45%, 71%, 80% and 99% Cr 6+ (initial metal concentration = 1 mM) was removed from the medium by Staphylococcus aureus K1 after 2, 4, 8, 16 and 24 h of incubation, respectively ( Figure 1).
Agronomy 2020, 10, x FOR PEER REVIEW 6 of 18 The bacterial strain K1, capable of tolerating a Cr concentration of up to 22 mM, was selected for further studies. Morphologically, it is characterized by Gram-positive cocci (≈1 μm) with yellowish golden color. Chemically, it is oxidase-and coagulase-negative and catalase-positive ( Table 2). The BLASTn investigation showed that it has a close resemblance (99%) to Staphylococcus aureus strain ATCC 12600 (NR_115606.1) and Staphylococcus aureus strain NBRC 100910 (MG971399.1). The similar 16S rDNA gene sequences from GenBank were used to carry out phylogenetic analysis, which also confirmed that the isolate K1 belongs to Staphylococcus aureus; therefore, it was named Staphylococcus aureus strain K1 (KX685332). This was done in order to remain confident that the bacterial strain used in this study was Staphylococcus aureus strain K1, as culture media can sometimes be contaminated with other bacteria. Gram-reaction +ve 4 Catalase +ve 5 Coagulase plasma reaction −ve

Effect of S. aureus K1 Contact Time on Chromium (Cr 6+ ) Reduction
Staphylococcus aureus K1 exhibited optimum growth at pH 8 and 35 °C. Under optimum growth conditions, the effect of contact time on bacterial ability to reduce the hexavalent Cr in the medium was observed. It was observed that the Cr reduction of S. aureus K1 increased with increasing contact time ( Figure 1). It was found that 26%, 45%, 71%, 80% and 99% Cr 6+ (initial metal concentration = 1 mM) was removed from the medium by Staphylococcus aureus K1 after 2, 4, 8, 16 and 24 h of incubation, respectively ( Figure 1).

Effect of S. aureus K1 on Plant Growth Promotion
Chromium stress substantially decreased the growth of wheat plants. A significant reduction in the length of shoots (31.18%), roots (32.02%) and spikes (40.70%) and the dry weight of shoots (34.29), roots (44.17) and grains (31.06%) of the plant was observed at 100 mg·kg −1 Cr concentration alone as compared to S. aureus K1 inoculated seeds + 100 mg·kg −1 Cr concentration ( Figure 2). A significant change in shoot and root length was observed in inoculated plants as compared to uninoculated plants at all levels of Cr. Wheat plants stressed with 50 mg·kg −1 of Cr showed an observable reduction in growth attributes; however, this decrease was minimized in inoculated plants compared to uninoculated plants, as shown in Figure 2. The growth was gradually decreased when the Cr concentration in the growth medium increased from 25 to 100 mgkg −1 (Figure 2A-D). Moreover, the maximum growth reduction was noticed with 100 mgkg −1 of Cr stress. The data

Effect of S. aureus K1 on Plant Growth Promotion
Chromium stress substantially decreased the growth of wheat plants. A significant reduction in the length of shoots (31.18%), roots (32.02%) and spikes (40.70%) and the dry weight of shoots (34.29), roots (44.17) and grains (31.06%) of the plant was observed at 100 mg·kg −1 Cr concentration alone as compared to S. aureus K1 inoculated seeds + 100 mg·kg −1 Cr concentration ( Figure 2). A significant change in shoot and root length was observed in inoculated plants as compared to uninoculated plants at all levels of Cr. Wheat plants stressed with 50 mg·kg −1 of Cr showed an observable reduction in growth attributes; however, this decrease was minimized in inoculated plants compared to uninoculated plants, as shown in Figure 2. The growth was gradually decreased when the Cr concentration in the growth medium increased from 25 to 100 mg·kg −1 (Figure 2A-D). Moreover, the maximum growth reduction was noticed with 100 mg·kg −1 of Cr stress. The data regarding plant growth attributes Agronomy 2020, 10, 1354 7 of 18 indicated that inoculation with S. aureus K1 significantly improved the wheat growth and dry biomass under Cr stress conditions. Agronomy 2020, 10, x FOR PEER REVIEW 7 of 18 regarding plant growth attributes indicated that inoculation with S. aureus K1 significantly improved the wheat growth and dry biomass under Cr stress conditions.

IRGA Parameters and Chlorophyll Contents
IRGA parameters such as transpiration rate, stomatal conductance and photosynthetic rate gradually reduced under increased Cr concentrations alone. The transpiration rate was greater at 25 mg kg −1 of Cr stress and decreased with increasing Cr stress levels at concentrations from 50 to 100 mg kg −1 . Without microbial inoculation, transpiration rate decreased by 12%, 21% and 32% under 25, 50 and 100 mg kg −1 Cr stress, respectively, as compared to control ( Figure 3A). Similarly, stomatal conductance and photosynthetic rate in uninoculated plants also reduced with increasing Cr concentrations. Stomal conductance decreased by 9%, 25%, 45% and photosynthetic rate decreased by 12%, 25% and 46% under 25, 50 and 100 mg kg −1 Cr stress, respectively, as shown in Figure 3B

IRGA Parameters and Chlorophyll Contents
IRGA parameters such as transpiration rate, stomatal conductance and photosynthetic rate gradually reduced under increased Cr concentrations alone. The transpiration rate was greater at 25 mg·kg −1 of Cr stress and decreased with increasing Cr stress levels at concentrations from 50 to 100 mg·kg −1 . Without microbial inoculation, transpiration rate decreased by 12%, 21% and 32% under 25, 50 and 100 mg·kg −1 Cr stress, respectively, as compared to control ( Figure 3A). Similarly, stomatal conductance and photosynthetic rate in uninoculated plants also reduced with increasing Cr concentrations. Stomal conductance decreased by 9%, 25%, 45% and photosynthetic rate decreased by 12%, 25% and 46% under 25, 50 and 100 mg·kg −1 Cr stress, respectively, as shown in Figure 3B On the other hand, as compared to untreated control, chlorophyll b was reduced by 15.36%, 27.27% and 40.80% in uninoculated wheat plants and by 14.44%, 27.24% and 40.63% in inoculated wheat plants under 25,50 and 100 mg·kg −1 Cr stress, respectively, as shown in Figure 3E. A gradual decrease in carotenoid contents was also observed in inoculated and uninoculated plants with increasing level of Cr stress, where inoculated plants showed 6%, 19% and 27% reduction in carotenoid contents while uninoculated plants showed 9%, 19% and 28% reduction under 25, 50 and 100 mg·kg −1 Cr stress, respectively ( Figure 3F) Agronomy 2020, 10,  Figure 3E. A gradual decrease in carotenoid contents was also observed in inoculated and uninoculated plants with increasing level of Cr stress, where inoculated plants showed 6%, 19% and 27% reduction in carotenoid contents while uninoculated plants showed 9%, 19% and 28% reduction under 25, 50 and 100 mg kg −1 Cr stress, respectively ( Figure 3F)

Estimation of EL, MDA and H2O2
A substantial increase in EL was noted in both roots and shoots of wheat plants under Cr stress, as shown in Figure 4A

Estimation of EL, MDA and H 2 O 2
A substantial increase in EL was noted in both roots and shoots of wheat plants under Cr stress, as shown in Figure 4A Figure 4A,B). There was a noticeable increase in MDA content of leaves, showing lipid peroxidation due to high level of Cr stress, as shown in Figure 4C,D. Maximum MDA contents were observed in leaves and roots of uninoculated plants under 100 mg kg −1 Cr stress as compared to their respective controls. However, inoculation with S. aureus K1 reduced MDA content in all the plants of varying level of Cr stress compared to uninoculated plants. Likewise, a gradual rise in H2O2 of wheat leaves was observed with increasing levels of Cr ( Figure 4E,F). Furthermore, a noteworthy decrease in H2O2 content was observed in S. aureus K1 inoculated plants, both Cr-stressed and control.  There was a noticeable increase in MDA content of leaves, showing lipid peroxidation due to high level of Cr stress, as shown in Figure 4C,D. Maximum MDA contents were observed in leaves and roots of uninoculated plants under 100 mg·kg −1 Cr stress as compared to their respective controls. However, inoculation with S. aureus K1 reduced MDA content in all the plants of varying level of Cr stress compared to uninoculated plants. Likewise, a gradual rise in H 2 O 2 of wheat leaves was observed with increasing levels of Cr ( Figure 4E,F). Furthermore, a noteworthy decrease in H 2 O 2 content was observed in S. aureus K1 inoculated plants, both Cr-stressed and control.

Effect of S. aureus on Antioxidant Enzyme Activities
The findings revealed that SOD activity in leaves and roots was significantly higher at the 25 mg·kg −1 Cr level but gradually decreased with increasing Cr levels, both in uninoculated and inoculated plants. SOD activity increased by 19.59%, 5.22% and 6.98% in uninoculated plant leaves and by 17.58%, 5.22% and 3.08% in uninoculated plant roots under 25, 50 and 100 mg·kg −1 Cr treatments, respectively. However, inoculation with S. aureus K1 enhanced the SOD activity by 24.71%, 9.64% and 3.51% in leaves and 20.83%, 9.49%, and 4.34% in roots under 25, 50 and 100 mg·kg −1 Cr, respectively ( Figure 5A,B). As compared to noncontaminated treatments (control), a decline in the CAT activity was observed under Cr contamination ( Figure 5C,D). Inoculation with S. aureus K1 provoked a substantial increase in the activity of the CAT enzyme in wheat leaves ( Figure 5C). CAT activity in roots also improved (114.31 Units·g −1 FW) under bacterial inoculation as compared to uninoculated plants (102.66 Units g −1 FW) at 25 mg·kg −1 Cr ( Figure 5D). Moreover, abridged CAT activity was noticed at the highest level of Cr stress (100 mg·kg −1 ); activity at this level was increased by 5.52% in leaves and 3.63% in roots for uninoculated plants, while inoculated plants showed increase of 5.06% in leaves and 1.37% in roots, as shown in Figure 5C,D. The POD activity substantially (p < 0.05) increased due to addition of Cr as compared to control ( Figure 5E,F). There was a noticeable reduction in POD activity in leaves under bacterial inoculation with S. aureus strain K1 (22.27%, 11.99% and 0.21%) as compared to uninoculated treatments (21.63%, 10.12% and 2.92%) ( Figure 5E). There was a substantial increase in the activity of the APX enzyme observed under Cr stress in wheat plants, as shown in Figure 5G,H. There was increase in APX activity in plant shoots and roots, with the maximum production occurring at the Cr concentration of 25 mg·kg −1 , and the APX activity decreased at the highest Cr level in the growth medium ( Figure 5G,H). Furthermore, the maximum APX activity was observed in roots without inoculation at Cr concentration of 25 mg·kg −1 , as shown in Figure 5H. The findings revealed that SOD activity in leaves and roots was significantly higher at the 25 mg·kg −1 Cr level but gradually decreased with increasing Cr levels, both in uninoculated and inoculated plants. SOD activity increased by 19.59%, 5.22% and 6.98% in uninoculated plant leaves and by 17.58%, 5.22% and 3.08% in uninoculated plant roots under 25, 50 and 100 mg kg −1 Cr treatments, respectively. However, inoculation with S. aureus K1 enhanced the SOD activity by 24.71%, 9.64% and 3.51% in leaves and 20.83%, 9.49%, and 4.34% in roots under 25, 50 and 100 mg kg −1 Cr, respectively ( Figure 5A,B). As compared to noncontaminated treatments (control), a decline in the CAT activity was observed under Cr contamination ( Figure 5C,D). Inoculation with S. aureus K1 provoked a substantial increase in the activity of the CAT enzyme in wheat leaves ( Figure 5C). CAT activity in roots also improved (114.31 Units·g −1 FW) under bacterial inoculation as compared to uninoculated plants (102.66 Units g −1 FW) at 25 mg·kg −1 Cr ( Figure 5D). Moreover, abridged CAT activity was noticed at the highest level of Cr stress (100 mg·kg −1 ); activity at this level was increased by 5.52% in leaves and 3.63% in roots for uninoculated plants, while inoculated plants showed increase of 5.06% in leaves and 1.37% in roots, as shown in Figure 5C,D. The POD activity substantially (p < 0.05) increased due to addition of Cr as compared to control ( Figure 5E,F). There was a noticeable reduction in POD activity in leaves under bacterial inoculation with S. aureus strain K1 (22.27%, 11.99% and 0.21%) as compared to uninoculated treatments (21.63%, 10.12% and 2.92%) ( Figure 5E). There was a substantial increase in the activity of the APX enzyme observed under Cr stress in wheat plants, as shown in Figure 5G,H. There was increase in APX activity in plant shoots and roots, with the maximum production occurring at the Cr concentration of 25 mg·kg −1 , and the APX activity decreased at the highest Cr level in the growth medium ( Figure 5G,H). Furthermore, the maximum APX activity was observed in roots without inoculation at Cr concentration of 25 mg·kg −1 , as shown in Figure 5H.

Cr Accumulation in Plants
The data regarding Cr accumulation in shoots and roots of the wheat plants are shown in Figure 6A,B. With increasing concentration of applied Cr, a gradual increase in Cr concentrations was observed in roots and shoots in a dose-additive manner. In addition, inoculation of S. aureus K1 significantly decreased the Cr concentrations both in shoots and roots as compared to uninoculated plants.

Cr Accumulation in Plants
The data regarding Cr accumulation in shoots and roots of the wheat plants are shown in Figure 6A,B. With increasing concentration of applied Cr, a gradual increase in Cr concentrations was observed in roots and shoots in a dose-additive manner. In addition, inoculation of S. aureus K1 significantly decreased the Cr concentrations both in shoots and roots as compared to uninoculated plants.

Cr Accumulation in Plants
The data regarding Cr accumulation in shoots and roots of the wheat plants are shown in Figure 6A,B. With increasing concentration of applied Cr, a gradual increase in Cr concentrations was observed in roots and shoots in a dose-additive manner. In addition, inoculation of S. aureus K1 significantly decreased the Cr concentrations both in shoots and roots as compared to uninoculated plants.

Discussion
The major objective of our research was to appraise the effectiveness of Staphylococcus aureus K1 treatment in reducing the toxic effects of Cr stress in wheat plants. An indigenous bacterial strain, Staphylococcus aureus K1 (GenBank accession no. KX685332), capable of tolerating up to 22 mM of Cr 6+ was isolated from a metal-polluted environment. Numerous research studies with similar metal-tolerant bacterial isolations from metal-contaminated sites have been reported [35,50,51]. Our results also supported the findings of Mustapha and Halimoon [52], who isolated a total of 21 isolates from electroplating industries and reported that merely 5 of them were Cr-tolerant (up to 50 mg·L −1 ). The results of the current study show that S. aureus K1 increased plant growth parameters under Cr metal stress (Figure 2).

Detoxification of Metals by S. aureus K1
Microbes have a number of metal resistance mechanisms involving chromosomes, transposon-encoded genes or plasmids. These mechanisms are mostly plasmid-facilitated and show resistance to some particular anion or cation [53]. Metals can have different impacts inside cells depending upon their concentration [53]; once a certain level is exceeded, bacteria respond with the initiation of a number of resistance mechanisms, including metallothioneins, P-type ATPases, CDF transporters and RND efflux pumps [54]. The genes located on plasmids, chromosomes or transposons that are responsible for resistance can easily be transferred to new community members from their point of location [53,55].
The genotype of bacteria, the nature and type of the metal and the pH of the culturing media are among the factors responsible for showing the degree of tolerance of microbes to various metals (Hg, Co, Pb, Ag, Zn, Mn, Cu, Cr) [56]. This kind of resistance against toxic heavy metals might be recognized by employing a number of potential methods like bioaccumulation of heavy metals by microbes, ion exclusion and low-molecular-weight binding protein production [57,58]. Elevated levels of metal resistance systems in bacterial cells are an indication of environmental heavy metal bioavailability [59]. The results of Chudobova et al. [60] showed a maximum resistance and capability of S. aureus strains under Cd 2+ and Zn 2+ ions. This resistance observed in S. aureus might be due to the efflux system containing a P-type ATPase transport system acting against Cd 2+ ions [53,61].

Effect of S. aureus on Plant Growth Promotion under Cr Metal Stress
Different wheat varieties may differ in their response to different concentration of Cr in the soil. This could be attributed to various biological aspects of wheat varieties, as different wheat varieties show differences in growth parameters (e.g., leaf size). A heavy metal like Cr can easily make its way

Discussion
The major objective of our research was to appraise the effectiveness of Staphylococcus aureus K1 treatment in reducing the toxic effects of Cr stress in wheat plants. An indigenous bacterial strain, Staphylococcus aureus K1 (GenBank accession no. KX685332), capable of tolerating up to 22 mM of Cr 6+ was isolated from a metal-polluted environment. Numerous research studies with similar metal-tolerant bacterial isolations from metal-contaminated sites have been reported [35,50,51]. Our results also supported the findings of Mustapha and Halimoon [52], who isolated a total of 21 isolates from electroplating industries and reported that merely 5 of them were Cr-tolerant (up to 50 mg·L −1 ). The results of the current study show that S. aureus K1 increased plant growth parameters under Cr metal stress (Figure 2).

Detoxification of Metals by S. aureus K1
Microbes have a number of metal resistance mechanisms involving chromosomes, transposon-encoded genes or plasmids. These mechanisms are mostly plasmid-facilitated and show resistance to some particular anion or cation [53]. Metals can have different impacts inside cells depending upon their concentration [53]; once a certain level is exceeded, bacteria respond with the initiation of a number of resistance mechanisms, including metallothioneins, P-type ATPases, CDF transporters and RND efflux pumps [54]. The genes located on plasmids, chromosomes or transposons that are responsible for resistance can easily be transferred to new community members from their point of location [53,55].
The genotype of bacteria, the nature and type of the metal and the pH of the culturing media are among the factors responsible for showing the degree of tolerance of microbes to various metals (Hg, Co, Pb, Ag, Zn, Mn, Cu, Cr) [56]. This kind of resistance against toxic heavy metals might be recognized by employing a number of potential methods like bioaccumulation of heavy metals by microbes, ion exclusion and low-molecular-weight binding protein production [57,58]. Elevated levels of metal resistance systems in bacterial cells are an indication of environmental heavy metal bioavailability [59]. The results of Chudobova et al. [60] showed a maximum resistance and capability of S. aureus strains under Cd 2+ and Zn 2+ ions. This resistance observed in S. aureus might be due to the efflux system containing a P-type ATPase transport system acting against Cd 2+ ions [53,61].

Effect of S. aureus on Plant Growth Promotion under Cr Metal Stress
Different wheat varieties may differ in their response to different concentration of Cr in the soil. This could be attributed to various biological aspects of wheat varieties, as different wheat varieties show differences in growth parameters (e.g., leaf size). A heavy metal like Cr can easily make its way to aerial portions of plants, where it will affect their shoot metabolism at the cellular level and cause severe damage to minerals, water and nutrients, consequently retarding plant growth [10,62].
However, bacterial inoculation may improve the nutritional requirements of both micro-(Mn, Zn, Cu and Fe) and macronutrients (N, P and K) by modifying host physiology, which results in changed uptake pattern of roots. Similarly, a recent investigation done by Islam et al. [63] showed an increase in Fe and K concentrations in maize plants under Cr stress due to bacterial inoculations. According to an observation, plants with bacterial inoculation showed a reduction in metal accumulation in their aerial parts, which might be due to delayed translocation of metals from roots to upper parts [64]. Similar observations were recorded in this current research. Moreover, we isolated S. aureus K1 from wastewater that was contaminated with Cr, so the microbes may have the capability of performing metal detoxification as a part of their metabolic system. There was substantial improvement in plant growth and leaf pigments due to inoculation of specific microbes [63].

Chlorophyll Contents
Higher chlorophyll contents were observed in plants with bacterial inoculation compared to uninoculated plants ( Figure 3). However, with further increasing metal concentrations, a reduction in chlorophyll contents was noted. This is in agreement with the findings of another research study, where chlorophyll a and chlorophyll b in wheat plants decreased with increasing concentrations of Pb in the growth medium [65].

ROS Species and Antioxidant Enzyme Production
Reactive oxygen species can be produced in plants when exposed to Cr 6+ , which may damage the photosynthetic apparatus and protein complex of thylakoid membranes and result in inhibition of chlorophyll production [66]. In adverse conditions, plants release MDA contents; this reveals the level of lipid peroxidation, as MDA is the last decomposition product of membrane lipid peroxidation [67]. The increase in MDA contents found in the present study is indicative of imbalance between the generation and removal of free radicals in the cells [68]. The decreased lipid peroxidation with S. aureus K1 inoculation under Cr stress could be due to the increase in ROS-scavenging enzyme production in plants. This may be supported by a previously published study which revealed that the gene profile of metal detoxifying enzymes was activated by bacterial inoculation to deal with metal stress [69]. Reactive oxygen species are generated in response to stress caused by heavy metals like hexavalent Cr, and plants have a detoxifying antioxidant enzyme system for their maintenance. These enzymes are POD, SOD, APX and CAT, and they work alongside other non-enzymatic antioxidants. The activities performed by antioxidant enzymes in plants under metal stress are extremely variable and dependent on plant species, metal concentration, metal ions and exposure time period [70]. At low metal concentration, SOD activity may increase, but it becomes constant with increased metal concentration [71]. The enhancement in CAT activity was also noted in a number of plants under metal stress [72]. An increase in CAT activity was also observed as an adaptive trait of isolate CPSB21 [73]. Increased antioxidant enzyme activities in plants with inoculation of CPSB21 may be due to increases in mRNA/gene expression of antioxidant enzymes as compared to uninoculated plants [74].

Reduction of Cr Concentration in Plants by Bacterial Inoculation
A significant difference was found between uninoculated and S. aureus K1 inoculated plants in terms of Cr concentration. In contaminated soil, the results showed that the level of Cr was higher in the roots of wheat plants than it was in the shoots, which may be due to decreased translocation of Cr from roots to shoots of plants [75,76]. Immobilization of Cr in root cell vacuoles may lead to higher Cr accumulation in roots, which can cause toxicity in plants [77]. In the present study, inoculation of wheat plants with Cr-resistant microbes decreased the Cr concentration and its translocation from soil to roots and upper parts of wheat plants. The reduction of hexavalent Cr (Cr 6+ ) to trivalent Cr (Cr 3+ ) by bacterial isolates may be the reason for the improved growth of wheat plants [78] and hence the decreased level of the Cr contents in soil. Hasnain and Sabri [79] also reported a pattern of decreased Cr uptake and accumulation in roots and shoots of wheat plants inoculated with Pseudomonas sp.
A decrease in Cr concentration in soil was observed after wheat plant harvesting. This decrease was recorded in uninoculated Cr-contaminated wheat plants as a result of increased accumulation and uptake of Cr in roots and shoots [80]. Such decrease may also be due to Cr 6+ reduction into Cr 3+ under the influence of bacterial inoculation [78,81]. Scientists are also considering the use genetically engineered microorganisms (GEM), which may be well adjusted to their local environment (both climatic and soil) for effective elimination of heavy metals from contaminated soils [58,82,83].

Conclusions
The outcomes of this study indicate that the application of the peat-moss-based microbial inoculum improved plant growth and yield parameters and comparatively decreased metal accumulation by the plants. Overall, gas exchange attributes and chlorophyll contents increased with S. aureus K1 inoculation. This research study concluded that S. aureus K1 reduced the toxicity of Cr in wheat plants. The Cr-resistant S. aureus K1 supported the plant growth, decreased and detoxified Cr in plants and allowed better production of wheat in a Cr-contaminated environment. However, in-depth exploration (i.e., at the molecular level) of the alleviative mechanisms in plants should be conducted in future studies.