*3.5. 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,B. Uninoculated wheat plants showed more EL in leaves and roots under all Cr levels (0, 25, 50 and 100 mg·kg<sup>−</sup>1) as compared to inoculated plants. EL in uninoculated leaves was increased by 17.98%, 36.40% and 56.52% and EL in uninoculated roots increased by 9%, 32% and 53% under 25, 50 and 100 mg·kg−<sup>1</sup> Cr, respectively. On the other hand, inoculation with *S. aureus* K1 increased EL in leaves by 15.83%, 33.26% and 55.90% and in roots by 13%, 33% and 56%, under 25, 50 and 100 mg·kg−<sup>1</sup> Cr, respectively (Figure 4A,B).

**Figure 4.** Influence of the different Cr levels (0, 25, 50, 100 mg·kg<sup>−</sup>1), with and without peat-moss-based microbial inoculation, on EL in leaves (**A**), EL in roots (**B**), MDA in leaves (**C**), MDA in roots (**D**), H2O2 in leaves (**E**) and H2O2 in roots (**F**) of wheat plants. Bars indicate the mean values and standard deviation of three replicates. Different bar letters show significant changes among various treatments at *p* < 0.05.

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−<sup>1</sup> 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.

#### *3.6. E*ff*ect 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−<sup>1</sup> 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−<sup>1</sup> 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−<sup>1</sup> 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−<sup>1</sup> FW) under bacterial inoculation as compared to uninoculated plants (102.66 Units g−<sup>1</sup> FW) at 25 mg·kg−<sup>1</sup> Cr (Figure 5D). Moreover, abridged CAT activity was noticed at the highest level of Cr stress (100 mg·kg<sup>−</sup>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<sup>−</sup>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<sup>−</sup>1, as shown in Figure 5H.

**ƌŽŶĐĞŶƚƌĂƚŝŽŶ;ŵŐŬŐͲϭͿ**

<sup>Ă</sup> <sup>Đ</sup> <sup>Ğ</sup>

**Figure 5.** Influence of the different Cr levels (0, 25, 50, 100 mg·kg<sup>−</sup>1), with and without peat-moss-based microbial inoculation, on SOD in leaves (**A**), SOD in roots (**B**), CAT in leaves (**C**), CAT in roots (**D**), POD in leaves (**E**), POD in roots (**F**), APX in leaves (**G**) and APX in roots (**H**) of wheat plants. Bars indicate the mean values with standard SD of three replicates. Different bar letters show significant changes among various treatments at *p* < 0.05.

#### *3.7. 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.

**Figure 6.** *Cont*.

**Figure 6.** Influence of the different Cr levels (0, 25, 50, 100 mg·kg<sup>−</sup>1), with and without peat-moss-based microbial inoculation, on Cr concentrations in shoots (**A**) and roots (**B**) of wheat plants. Bars indicate the mean values with standard SD of three replicates. Different bar letters show significant changes among various treatments at *p* < 0.05.

#### **4. 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 Cr6<sup>+</sup> 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<sup>−</sup>1). The results of the current study show that *S. aureus* K1 increased plant growth parameters under Cr metal stress (Figure 2).

#### *4.1. 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 Cd2<sup>+</sup> and Zn2<sup>+</sup> ions. This resistance observed in *S. aureus* might be due to the efflux system containing a P-type ATPase transport system acting against Cd2<sup>+</sup> ions [53,61].

#### *4.2. E*ff*ect 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].

#### *4.3. 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].

#### *4.4. ROS Species and Antioxidant Enzyme Production*

Reactive oxygen species can be produced in plants when exposed to Cr6+, 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].

#### *4.5. 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 (Cr6<sup>+</sup>) to trivalent Cr (Cr3<sup>+</sup>) 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 Cr6<sup>+</sup> reduction into Cr3<sup>+</sup> 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].
