**1. 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 plants [3], animals and humans [4]. In agricultural systems, Cr can easily move to different parts of crops and accumulate there to be later consumed by the animals and humans [5]. Soil contamination by Cr and other heavy metals impacts biodiversity negatively and badly disturbs the living entities in the soil [6].

Major origins of Cr contamination are the leather industry [7,8], mining [1], steel industry, paint industry, wood preservatives, volcanic eruption and weathering [9]. Chromium exerts negative effects on plants by reducing the plant height and root growth, interrupting the germination process, causing disproportion in nutrient levels, exerting harmful effects on photosynthesis, retarding soil microbial activities, inhibiting enzyme activity and stimulating the formation of reactive oxygen species (ROS) which result in induction of oxidative stress in plants [1,10,11]. Chromium can cause different malfunctions in human biological systems that may lead to the death of affected persons [12–14]. Wastewater effluents from the industries are discharged directly into water bodies that are utilized mostly for irrigation purposes. Farmers have to rely on this untreated contaminated water due to limited resources and inadequate sanitation facilities [15].

A major staple food across the world is wheat (*Triticum aestivum* L.), which fulfils food requirements of about 50% of the worldwide population [16]. Amongst wheat-producing countries, Pakistan comes ninth in the world. Wheat subsidizes necessary amino acids, vitamins and minerals, dietary fibers and phytochemicals in our diet [17]. Wheat can accumulate higher Cr concentration in stems followed by leaves and grains [18]. According to the literature, increased heavy metal accumulation in wheat tissues has become a potential source of food chain contamination that can cause serious abnormalities to human biological systems [19,20]. Crops may also have the ability to reduce the Cr from Cr6<sup>+</sup> to Cr3. This reduction process is likely to happen in roots as a detoxification mechanism [21]. There are a number of remediation methods used to treat sites contaminated with toxic metals. Presently, scientists have made rampant use of biologically centered techniques to deal with such toxic contaminants in order to remove them from environmental entities, including water, air and soil, or at least make them less damaging to the ecosystem [22]. The phytoremediation technique is a modernized method with a lower budget and environmentally sustainable system [23]; it destroys contaminants by using plants along with their rhizospheric microorganisms. Microbial-assisted phytoremediation helps to deal with toxic heavy metals by stabilizing or transforming them to less toxic forms in carrier materials such as soil, shallow water, sediments or groundwater [24]. Microbes have the capability to modify their genetic sequences in response to variation in environmental factors [25]. In soil polluted with heavy metals, microbes assist the plants by producing various growth-regulating substances, such as organic acids, hormones, siderophores and enzymes, that help in plant growth promotion by involving diverse mechanisms, namely acidification, precipitation, redox reactions and chelation [26]. Likewise, roots excrete beneficial nutrients to support the successful colonization and growth of microbes [26]. Chromium-reducing bacteria have the capability to remediate Cr toxicity by reducing Cr6<sup>+</sup> into Cr3<sup>+</sup> in the rhizosphere through bioaccumulation and biosorption mechanisms [27]. *Staphylococcus aureus* is a Gram-positive, ubiquitous and round-shaped facultative anaerobe that grows in clusters, forming a biofilm on surfaces. It can grow in a range of growth temperature from 7 to 48 ◦C, with 37 ◦C as the optimal temperature for growth [28]. It was isolated from tannery effluent and characterized as a chromium-reducing bacterium. The application of phytoremediation, along with Cr-resistant bacteria for detoxification of Cr6<sup>+</sup>, has been considered a safe, effective and economical technique over customary techniques [29,30]. In this study, the alleviative role of *Staphylococcus aureus* strain K1 under Cr stress was evaluated in wheat plants. It was hypothesized that microbes (such as *Staphylococcus aureus* strain K1) may alleviate Cr toxicity in wheat by enhancing antioxidant enzymatic activities of wheat while reducing oxidative stress through biotransformation (Cr6<sup>+</sup> into Cr3<sup>+</sup>) and biosorption of Cr.

#### **2. Materials and Methods**

#### *2.1. 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 K2Cr2O7 in the laboratory, and soil was spiked with final Cr concentrations of 0, 25, 50 and 100 mg·kg−<sup>1</sup> 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.


**Table 1.** Soil characterization of pot experiment.

#### *2.2. 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<sup>−</sup>1, 10<sup>−</sup>2, 10<sup>−</sup><sup>3</sup> and 10<sup>−</sup>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 Cr6+. 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 Cr6<sup>+</sup> [35]. The bacteria

that showed maximum resistance to the highest concentration of hexavalent Cr were selected for use in further studies.

#### *2.3. 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].

#### *2.4. 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 × 108 CFU mL−<sup>1</sup> [39].

#### *2.5. 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 (H2O2) for 30 min [40]. The sterilized seeds were immersed in double volume of bacterial suspension (1 × 10<sup>8</sup> CFU mL<sup>−</sup>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.

#### *2.6. Treatments*

The experiment was conducted in plastic pots using different concentrations of Cr (0, 25, 50 and 100 mg·kg<sup>−</sup>1) in the presence and absence of bacterial inoculation. Different treatments were as follows: T1 (Control), 0 mg·kg−<sup>1</sup> Cr; T2, 25 mg·kg−<sup>1</sup> Cr; T3, 50 mg·kg−<sup>1</sup> Cr; T4, 100 mg·kg−<sup>1</sup> Cr; T5, 0 mg·kg−<sup>1</sup> Cr + *S. aureus* K1; T6, 25 mg·kg−<sup>1</sup> Cr + *S. aureus* K1; T7, 50 mg·kg−<sup>1</sup> Cr + *S. aureus* K1; T8, 100 mg·kg−<sup>1</sup> Cr + *S. aureus* K1.

#### *2.7. 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.

#### *2.8. 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).
