Unravelling Potential of Silicon and Rhizobacteria in Reducing Cd-related Health Risk in Grazing Animals by Enhancing Maize Fodder Quality

Unravelling Potential of Silicon and Rhizobacteria in Reducing Cd-related Health Risk in Grazing Animals by Enhancing Maize Fodder Quality | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Unravelling Potential of Silicon and Rhizobacteria in Reducing Cd-related Health Risk in Grazing Animals by Enhancing Maize Fodder Quality Seemal Naeem, Waqas khan, Tahmina Nazish, Muhammad Awan, Usman Ijaz, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6592002/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Cadmium (Cd) enrichment in agricultural soils due to wastewater irrigation poses significant risks to the growth, quality, and yield of maize fodder. This study explores the role of SN215 and silicon (Si) in mitigating cadmium (Cd) toxicity in maize fodder and reducing associated health risks in grazing animals. The SN215 strain, isolated from the wheat rhizosphere and identified as SN215, exhibited 83% Cd biosorption efficiency at a medium Cd concentration (10 ppm). Under controlled greenhouse conditions, the combined application of SN215 and Si significantly enhanced maize growth, resulting in a two-fold improvement in shoot fresh and dry biomass. Furthermore, the treatment improved relative water content (RWC), phenolic levels, chlorophyll concentrations, and protein content, restoring RWC to 60% and increasing phenolic content by 10% in comparison to plants under Cd-only stress. The treatment significantly increased antioxidant enzyme activities (ascorbate peroxidase, catalase, superoxide dismutase, and peroxidase) while reducing oxidative stress markers like malondialdehyde and hydrogen peroxide by 61.96% and 59.43%, respectively. Moreover, the combined application of SN215 and Si reduced Cd uptake in shoots by 95% and soil Cd levels by 30%. Health risk assessments revealed a negligible daily intake of metals and a health risk index for grazing animals with SN215 and Si treatment, highlighting its effectiveness in mitigating Cd toxicity. The findings demonstrate the potential of SN215 and Si co-application as an eco-friendly strategy to improve fodder quality and reduce health risks in Cd-contaminated environments. Cleaner production Sustainability Wastewater Animal fodder Cd stress Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Wastewater application in the agriculture sector of developing countries has been gaining attention in recent years due to freshwater scarcity (FAO, 2023 ; Yerli et al., 2024 ). Wastewater or industrial effluents contain toxic and heavy metal ions (even after recycling), which ultimately deteriorate the fodder yield and quality (Wen et al., 2018 ; Islam et al., 2023 ). Heavy metals in wastewater slow plant growth by creating reactive oxygen species (ROS) or by changing the functions of important enzymes by taking away metals and nutrients that plants need (Zhu et al., 2021 ; Alsafran et al., 2023 ; Hu et al., 2023 ). For instance, Cadmium (Cd) enrichment in several soils across the globe was linked with the use of Cd rich industry effluent or wastewater for irrigation purposes (Rezapour et al., 2024 ). The Cd ranked seventh among potential hazardous elements with significant health and environmental health related hazards (Genchi et al., 2020 ). Cd enriched fodder crops showed reduced fodder quality, such as reduced growth and increased electrolyte leakage and enhanced production of ROS in Sorghum bicolor (Hassan et al., 2020), reduced plant height as well as biomass in Medicago sativa (Kareem et al., 2023 ), reduced shoot and root length, dry and fresh weight, and chlorophyll content in Zea mays (Shah et al., 2023 ), decreased protein as well as lipid content in Oryza sativa (Lan et al., 2023 ), and amount of carbohydrates and their conjugates in Panax notoginseng (Lu et al., 2023 ). Studies have shown that several animals' consumption of Cd-rich fodder is a major source of Cd toxicity (Sapunar-Postrunik et al., 2001). A significant accumulation of Cd has been reported in faeces, hair, and blood samples of cows and buffalos fed with Cd-contaminated forage crops. Residents exposed to the milk and meat of these cattle showed serious health concerns (Hussain et al., 2022 ). Maize, being one of most important cereal crops, is susceptible to cadmium stress and maize products safety is directly linked to food safety as Cd can be transferred from fodder to animals and then to humans, hence being a potential threat to both animals and humans (Liu et al., 2015 ; Lamb et al., 2016 ; Rizwan et al., 2017 ). The accumulation of Cd at high concentrations in maize tissues reduces shoot growth, plant height, plant fresh and dry weight, activity of antioxidant enzymes, chlorophyll content and ultimately, poor fodder quality (He et al., 2021 ; Ruiz-Huerta et al., 2022 ). It was found that Cd decreased the yield of fodder crops by stopping roots from growing longer, messing up nutrient uptake, stopping reproductive development, slowing down photosynthesis, and ultimately lowering yield in higher plants (Anjum et al., 2016 ; Liu et al., 2020 ). At the cellular level, Cd induces oxidative stress, destroying the cellular structures which leads to cellular death (Clemens et al., 2013 ; Shanying et al., 2017 ). Moreover, Cd induced greater oxidative damage and accumulation of malondialdehyde (MDA) concentration in the shoot and leaf of fodder crops was associated with a decline in fodder quality (Ahmad et al., 2016 ; Pandian et al., 2020 ; Awan et al., 2023 ). This prompts an examination of methods for reducing Cd toxicity in fodder crops, particularly maize fodder. Examining approaches to reduce Cd uptake and its translocation from root to shoot could provide the answer, leading to an improvement in maize fodder yield and quality. One can argue that developing tolerant crop varieties using gene editing techniques could be a more productive solution. However, the use of gene editing techniques is greatly influenced by expert personnel, plant species, and experimental conditions, thus indicating the adoption of agronomic techniques as a green technology for ameliorating Cd toxicity in fodder crops. Traditional physicochemical methods using hydrated lime for neutralization of soil Cd and soil replacement for Cd remediation are highly effective, but their applications are limited due to high costs and soil pollution (Lim et al., 2013 ; Bian et al., 2016 ; Kumpiene et al., 2019 ; Mu et al., 2024 )). Therefore, there is an urgent need to develop innovative techniques to sustain maize fodder production and mitigate Cd toxic effects. When it comes to cleaning up metal-contaminated farmland, biological methods using different types of plant growth-promoting rhizobacteria (PGPR) have gotten much attention (Zafar-Ul-Hye et al., 2018 ; Khanna et al., 2019 ; Ahmed et al., 2024). PGPRs are very good at keeping heavy metals in place and stopping them from moving around in plants by forming complexes, precipitating them, and absorbing them (Khanna et al., 2019 ). Researchers have extensively studied the PGPR's inherent metal immobilization capabilities and metal resistance to promote plant growth and reduce metal accumulation in plants. Moreover, metal-resistant bacteria release growth-promoting substances in plants' rhizosphere, such as solubilized phosphates, siderophores, and indole-3-acetic acid, to confer tolerance in plants under a metal-contaminated environment (Kumar et al., 2023; Ijaz et al., 2024 ). Therefore, this study examined the role of SN215 in reducing Cd toxicity in maize fodder. Shoot development is one of the most important plant traits in fodder crops, as higher shoot growth and development produce higher fodder yield with better fodder quality traits (Prajapati et al., 2023 ). In this context, Salicilic acid (Si) is important for its critical role in improving shoot growth, rigidity, and stress tolerance (Mir et al., 2022 ; Ali and Bijay, 2024 ). The Si application improved shoot growth by improving nutrient uptake, which later improved shoot length, shoot fresh weight, shoot relative contents and shoot greenness (higher chlorophyll contents) (Akhter et al., 2023 ) Moreover, the beneficial role of Si in conferring Cd tolerance was associated with (i) protecting ROS induced cell death (Xuebin et al. 2020 ), (ii) higher chlorophyll contents (Yan et al., 2023 ), and (iii) higher cellular turgidity and elasticity (Jan et al., 2018 ). The application of Si was also examined for conferring Cd toxicity in maize fodder. Moreover, the combined application of Si was examined for improving maize fodder yield and Cd related health risks in grazing animals. Hence, a study was conducted to evaluate the combined role of Si and Lysinibacillus sp. in mitigating the Cd toxicity in maize fodder. Furthermore, we tend (i) to examine the synergistic impact of Si and Lysinibacillus sp. in improving growth parameters and fodder quality traits, such as chlorophyll content, protein levels, and water retention under Cd stress (ii) the effectiveness of Si and Lysinibacillus sp. in reducing oxidative stress markers (e.g., MDA, H₂O₂) and enhancing the antioxidant defense system in maize fodder under Cd stress; lastly, (iii) to analyze the potential of Si and Lysinibacillus sp. to limit Cd uptake/ its bioavailability in maize fodder shoots and assess the health risk index (HRI) for grazing animals consuming Cd-contaminated fodder. 2. Materials and Methods 2.1. Isolation and Identification of Rhizobacteria Soil samples were collected from the rhizosphere of wheat crops in South Punjab, Pakistan, and stored in plastic bags at 4°C until further analysis. Serial dilutions (10⁻¹ to 10⁻⁹) were prepared by taking 10 g of rhizosphere soil. Microorganisms from each dilution were then cultured on petri plates, which were incubated at 37°C for 24 hours. After incubation, colonies of varying sizes and shapes were observed on the media, representing mixed cultures. To isolate pure colonies, each colony was streaked onto separate petri plates. Repeated streaking was performed to purify the bacterial isolates (Loutfi et al., 2020). These purified cultures were incubated again at 37°C for 24 hours. DNA was extracted from the 24-hour-old cultures following the optimized method of Sambrook et al. ( 1989 ) and stored in double-distilled deionized water at -20°C for further analysis. The 16S rRNA gene was amplified using a set of universal primers, fD1 and rD1, as previously described (Weisburg et al., 1999). The samples were sent to company for sequencing 16s RNA gene. ClustalW was used to perform multiple sequence alignment of the 16S rRNA gene sequence with its homologous sequences. The phylogenetic tree was constructed using MEGA (v.11.0) software, using the Maximum Likelihood (ML) method with 1000 bootstrap replicates to measure evolutionary relationships. 2.2 Cd biosorption and Characterization of Bacterial Isolate Gram staining was performed to determine the bacterial characteristics. Morphological characterization, including the examination of cell shape and colony structure, was conducted using a compound microscope (model XSZ107BN-A11.1007-17, China). The biosorption of total Cd was investigated by inoculating pure bacterial isolate W2 (Table 1 ) into broth media. To assess the efficiency of SN215 in capturing cadmium (Cd), three different levels of Cd were used: low (5 ppm), medium (10 ppm), and high (15 ppm). A 1 mL volume of overnight bacterial cultures grown on petri plates was introduced into 100 mL of sterilized broth using 15 mL falcon tubes. The tubes were incubated at 30°C for 24 hours. After incubation, the bacterial cultures were centrifuged at 13,000 revolutions per minute (rpm) for 10 minutes. The supernatants were collected to measure the remaining Cd content using a multi-sequential AAS (iCE 3000 SERIES) against prepared standards of known concentration. Then, Cd removal efficiency of bacterial isolate was also measured using the following formula. Table 1 Pollution load index, daily intake, and health risk assessment of Cd in animals in treatments Treatments Pollution Load Index (PLI) Daily Intake of Metal (DIM) Health Risk Index (HRI) Cow Sheep Buffalo Cow Sheep Buffalo T1 0.000 0 0 0 0 0 0 T2 0.000 0 0 0 0 0 0 T3 0.000 0 0 0 0 0 0 T4 0.000 0 0 0 0 0 0 T5 0.037 0.003 0.003 0.004 0.221 0.192 0.252 T6 0.013 0.002 0.002 0.002 0.156 0.134 0.176 T7 0.015 0.000 0.000 0.000 0.004 0.003 0.002 T8 0.009 0.000 0.000 0.000 0.006 0.005 0.006 R = (P0 – Pe / P0) *100 (1) In this equation, R = the percentage of metal removal by the fungal biomass, P0 = the initial concentration of metal ions (ppm) and Pe = the final concentration of metal ions (ppm) in the experimental media. 2.3 Experiment Setup and Climate Conditions The botanical garden of Government College University in Lahore hosted a greenhouse study. According to the Pakistan Meteorological Department (PMD), the region received an annual rainfall of 628 mm, with a maximum average temperature of 40°C and a minimum average temperature of 27°C. Zea mays L. (Maize) seeds were sown in pots, and after 10 days of growth, urea and diammonium phosphate (DAP) were applied. Si was used as a Si source. Similarly, 8 treatments were included in the experiment: T1 = Control; T2 = 2mM Silicon T3 = Bacteria; T4 = 2mM Silicon + Bacteria; T5 = Cd (10ppm); T6 = 2mM Silicon + Cd (10ppm); T7 = Bacteria + Cd (10ppm); T8 = 2mM Silicon + Bacteria + Cd (10ppm). Values were the average of three replicates ± standard error (SE). These treatments were applied in pots with 3 kg of soil. The physicochemical characteristics of Bhal soil were [Soil texture (Clay loam), sand (38%), silt (32%), clay (36%), organic matter (0.86%) (Estefan et al., 2013), pH (7.48) (Walkley et al., 1947), EC (1.37 dSm − 1 (Hailegnaw et al., 2019 ), and CEC (5.96 cmol Kg − 1 ) (Robertson et al., 1999)]. Six to seven seeds were sown in each pot, and approximately five to six seeds germinated after 4 to 10 days. The plants were harvested after 40 days. Roots and shoots were carefully removed and washed with distilled water to remove soil particles. Both plant and soil samples were stored in labelled zipper bags and immediately stored for further analysis. 2.4 Fodder Quality Properties 2.4.1 Measurement of Relative Water Content and Chlorophyll Relative water content (RWC) was measured by cutting a small piece of leaf. The fresh weight of the leaf was recorded, after which it was placed in a beaker filled with water for 4 hours. Following this, the leaf was reweighed to determine its turgid weight. The leaf was then placed in an oven for 24 hours to obtain its dry weight. RWC was calculated using the following formula: RWC = (FW- DW) / (TW-DW) FW = leaf fresh weight DW = leaf dry weight TW = leaf turgid weight Chlorophyll content was determined using the method described by Strain et al. (1996). Fresh leaves 0.1 grams were ground with 80% acetone in a pestle and mortar. After grinding, we used acetone to increase each sample's volume to 10 mL. The samples were then centrifuged for 5 minutes at 4000 rpm. The absorbance of the supernatant was measured using a UV/Visible spectrophotometer (Spectro scan 80D, Kyoto, Japan) at wavelengths of 663 nm and 645 nm to determine chlorophyll content. 2.4.2 Determination of Secondary Metabolites Total phenolics were measured using the method described by Sultana et al. (2012). To determine total phenolics, 0.1 g of shoot tissue was ground with 4 mL of acetone. A small amount (60 µL) of the sample extract was mixed with 4,740 µL of distilled water, 300 µL of Folin-Ciocalteu reagent, and 900 µL of Na 2 CO 3 . The prepared mixture was incubated at 55°C, then cooled, and its absorbance was measured using a UV/Visible spectrophotometer (Shimadzu UV 1201, Kyoto, Japan) at 760 nm. Protein content was determined using the Bradford ( 1976 ) method. Briefly, 0.1 g of shoot tissue was ground in a pestle and mortar. A 200 µL aliquot of each sample was diluted with 1800 µL of distilled water, followed by the addition of 2 mL of Bradford reagent. The mixture was then incubated for 15–20 minutes at 80°C. After incubation, absorbance was measured at 595 nm using a UV/Visible spectrophotometer (Spectro scan 80D, Kyoto, Japan). 2.4.3 Measurement of MDA, ROS Production and Antioxidant Enzyme Assay MDA (malondialdehyde) content was estimated using the method of Kanwal et al. ( 2018 ). Shoots (0.5 g) were ground with 5 mL of 20% trichloroacetic acid (TCA) solution. The mixture was centrifuged (HERMLE Z167M) at 10,000 rpm for 10 minutes. 2.5 mL of the supernatant, 1 mL of 0.5% (w/v) TCA and thiobarbituric acid (TBA) were added. This mixture was heated for 30 minutes at 95°C and then cooled in an ice bath. The absorbance was measured at 532 nm and 600 nm using a spectrophotometer (Spectro scan 80D, Kyoto, Japan). MDA content was calculated as the difference in absorbance between 532 nm and 600 nm using Lambert's equation. Hydrogen peroxide (H₂O₂) was determined by grinding 0.5 g of fresh maize shoots with 5 mL of 0.1% TCA solution, followed by centrifugation at 12,000 rpm for 15 minutes. After centrifugation, 1 mL of supernatant was mixed with 1 mL of 10 mM potassium phosphate (K-P) buffer (pH 7.0) and 2 mL of 1M potassium iodide (KI) solution. The absorbance of the mixture was measured at 390 nm to calculate the H₂O₂ concentration (Latef et al., 2019 ). Fresh maize shoots (0.6 g) were ground up in a buffer solution (0.2 M potassium phosphate, pH 7.8) that also had 0.1 mM EDTA in it. This was used to make plant extracts. The mixture was centrifuged (HERMLE Z167M) at 15,000 rpm for 20 guaiacol solution and 30 µL of 12 mM H₂O₂. The absorbance was checked at 436 nm using a spectrophotometer (Shimadzu UV-1201, Kyoto, Japan) (Sharma et al., 2017). 2.4.3. Maize fodder yield and Cd tolerance ability Fresh maize shoots were washed with distilled water to remove dust and soil particles. The shoots and roots were air-dried for 48 hours and then oven-dried at 70°C to achieve a constant weight. The weight of each sample was recorded. To find out how much cadmium (Cd) was in the shoots and roots, 0.5 g of maize samples were broken down in a dilute acid solution of 3 parts HNO 3 and 1 part HClO 3 at 180°C until clear fumes came out. Following the digestion process, we added distilled water to reach a total volume of 50 mL and then double-filtered the mixture to obtain a clear solution. Standards for Cd were prepared according to Jones et al. (1990), and the Cd concentration was measured using a multi-sequential Atomic Absorption Spectrophotometer (iCE 3000 SERIES). For soil samples, 1 g of soil was digested using the same di-acid mixture (HNO₃: HClO₄, 3:1) at 180°C until clear fumes were formed. After digestion, distilled water was added to make the final volume 50 mL, followed by double filtration to obtain a clear solution. Standards of Cd were prepared, and the concentration of Cd in soil samples was determined using the same multi-sequential Atomic Absorption Spectrophotometer (iCE 3000 SERIES) (Jones et al., 1990). 2.5 Health risk assessment for grazing animals 2.5.1 Pollution load index It is used to determine the contamination of heavy metals in soil. Its formula given by Liu et al. (2005) is: PLI = Metal concentration analysed in soil samples /Metal reference value in soil The reference value for Cd is 70 mg/kg (Singh et al. 2010 ). 2.5.2 Daily intake of metals The formula to find the daily intake of metals (DIM) is: DIM = Analysed metal concentration in forage*Conversion Factor*Daily food intake/Average body weight of animals For the conversion factor, value was 0.085 (Jan et al. 2010 ). For cows, daily intake of food was 12kg, and average body weight is 200 kg, whereas the daily intake and average weight of sheep were 1.3kg and 75kg, respectively (Johnsen and Aaneby 2019 ). For buffaloes, average weight was 300 kg, and their daily intake was 12.5kg (Briggs and Briggs, 1980). 2.5.3 Health risk index Oral reference dose for Cd is 0.017 mg/kg/day (USEPA 2020), and the formula to find the HRI is: HRI = Daily intake of metal/Oral reference dose 2.6 Statistical Analysis This experiment was performed in a completely randomized design and one way ANOVA by using Microsoft Excel 2013® (Microsoft cooperation, USA) and Statistix 8.1® (Analytical software, Tallahassee, USA), XL STAT for principal component analysis (PCA), was applied to analyze the results. MEGA 11 was used to prepare phylogenetic trees. The least significant design (LSD) was applied to compare the means of two separate groups. The results showed the mean of three replicates (n = 3) with standard error ± (SE). 3. Results 3.1. Isolation and Identification of Rhizobacteria In this study, bacteria were isolated from rhizosphere of wheat ( Triticum aestivum ) and subjected to morphological analysis and characterization (Supplementary Table 1). The bacterial strain known as SN215, which showed a probable potential for metal resistance, was gram positive. Taxonomic classification of the mentioned strain was done using sequence homology analysis and phylogenetic evaluation. A Basic Local Alignment Search Tool (BLAST) analysis was performed on 16S rRNA sequence and classified SN215 as SN215. To further confirm this homology, the relevant sequences from similar strains were retrieved from The National Center for Biotechnology Information (NCBI) database, and a phylogenetic tree was constructed using MEGA X, which demonstrated that SN215 clustered in close proximity to SN215 (Fig. 1 ). The strain was sequenced on the bases of 16S rRNA gene, and sequence was successfully submitted to GenBank under the accession ID SUB13767386 with the nucleotide accession number OR453182. 3.2. Biosorption Trial of SN215 Our results indicated that SN215 exhibited the highest biosorption efficiency 83%, at medium cadmium concentration. Based on these findings, the medium Cd concentration was selected for further investigation in pot experiments (Fig. 1 ), where the performance of bacteria in enhancing Cd tolerance of maize fodder was evaluated. 3.3 Maize Fodder Growth and Yield The combined application of bacterial strain SN215 and Si prominently improved the shoot biomass under Cd stress (Fig. 3 ). Our data depicted that plants of T8 demonstrated a remarkable increase in the shoot fresh biomass (two-folds) and shoot dry biomass (two-folds) compared T5 (Fig. 3 ). However, the applied Cd stress significantly influenced the growth parameters. T5 significantly reduced the shoot fresh weight (SFW) of maize (45.38%) compared to T1. A similar trend was observed for shoot dry weight of maize plants (Fig. 3 ). Moreover, T5 significantly (one-fold) reduced the shoot dry weight (SDW) of maize compared to T1. It is obvious from the figure that, irrespective of Cd toxicity in soil, different applied treatments enhanced the morphological traits of plants (Fig. 3 ). Maximum increase (two-folds) in SFW was reported with treatment T8 relative to T5. Similarly, the SDW of maize plant for Cd toxicity also showed a similar trend with maximum SDW up to (two-fold) in treatment T8 as compared to T5 (Fig. 3 ). 3.4 Maize Fodder Quality RWC exhibited significant variation across all treatments (Fig. 4 A). The RWC of maize was significantly reduced (29%) under 10 ppm Cd as compared to control plants. Remarkably, the combination of Si and SN215 under cadmium stress (T8) helped to restore RWC to around 60%, mitigating the negative impact of cadmium stress by approximately 16% compared to T5. Total phenolic content varied significantly under different treatments (Fig. 4 B). The control plants (T1) exhibited phenolic levels of about 85 µg g⁻¹, while plants receiving the combined treatment of Si + SN215 had the highest phenolic content of 53% increase over the control. Under 10 ppm Cd stress, the phenolic content decreased to 23% compared to T4. However, plants treated with both Si and SN215 under Cd stress maintained phenolic content at 10% recovery compared to T5. The greenness of maize fodder varied significantly under different treatments (Fig. 4 C). The highest chlorophyll content was recorded in T8, showing six-folds, nine-folds increase in chlorophyll a and chlorophyll b, respectively, as compared to T5. 10 ppm Cd stress led to a significant decline in chlorophyll content of 89%, 92.8% reduction in chlorophyll a and chlorophyll b respectively compared to T1. Total protein content exhibited a substantial variation among treatments (Fig. 4 D). T1 showed a protein concentration of approximately 18 mg g⁻¹. 10 ppm Cd stress led to a significant decline in protein content of 60% reduction compared to T1. Impressively, plants of T8 showed 67% increase in protein concentration compared to T5. 3.4.1 Cd Tolerance Capacity of Maize Fodder Application of Cd stress significantly increased the maize plant's oxidative stress damage, as measured by the level of MDA (Fig. 5 A). T5 reported a maximum increase in shoot MDA (up to one-fold) compared to T1. However, the co-application of Si and SN215 treatment decreased the shoot oxidative stress under Cd stress as measured by oxidative stress marker MDA. A maximum decrease (61.96%) in shoot MDA was reported with treatment T8 relative to T5 (Fig. 5 A). Moreover, application of Cd stress also increased H 2 O 2 concentration in maize plants, adding to oxidative stress burden of treated plants. Compared to control plants, T5 showed a maximum five-fold increase in shoot H2O2 concentration. However, conjoined application of Si and SN215 decreased the shoot oxidative stress against the increasing levels of Cd toxicity in soil. T8 reported the maximum decrease (59.43%) in shoot H2O2 concentration (Fig. 5 A). Our results clearly demonstrate that combined application of Si and SN215 predominantly alleviated oxidative stress of maize plants under Cd stress, conferring tolerance stress-treated plants against Cd. Data on the activities of antioxidant enzymes revealed that presence of Cd stresses significantly inhibited the functioning of APX and CAT in treated plants compared to control ones (Fig. 4 B). Under 10 ppm Cd toxicity, the concentrations of shoot APX (88.2%) and CAT (67%) were significantly lower than the control. When compared with T1, a three-fold increase in shoot APX as well as CAT was obvious when maize plants were subjected to Si + SN215, under 10 ppm Cd stress (Fig. 4 B), demonstrating stimulatory effect of Si and SN215 combination on activities of these antioxidant enzymes. The concentrations of shoot SOD (67.11%) and shoot POD (79.18%), among other pillars of antioxidant enzymes, were significantly lower under Cd stress compared to control. However, the application of Si and SN215 enhanced the concentrations of mentioned enzymes in maize plants under Cd toxicity. Maximum increase was observed in shoot SOD (71%) and shoot POD (three- fold) with T8 as compared to T5 (Fig. 4 C). 3.4.2. Cd Contents in Soil and Shoots of Maize Fodder In control plants, Cd uptake was negligible, while it peaked at 2.6 mg g⁻¹ DW in T5 (Fig. 6 A), marking a 13-fold increase compared to the T1. In T6, Cd uptake was reduced by 30%, while in T7, it was lowered by 42%. Notably, in T8 Cd uptake decreased by 95% compared to T5 (Fig. 5 A). Regarding soil Cd concentration, the concentration remained low in the non-Cd-stressed treatments (T1-T4), but it was maximum in the presence of Cd stress (T5) (Figs. 6 B). T8 led to a decrease in Cd toxicity to 30% as compared to T5, demonstrating its effectiveness in decreasing the bioavailability of Cd in the soil. The proposed study examined translocation factors (TF) and bioconcentration (BCF) (Fig. 6 ). This figure shows that overall Cd (TF) concentrations were less than 1, effectively preventing TF from root to shoot. These results showed that the roots of maize plants acquire higher total Cd ions than the shoots. The influence of maize plant capillary action on the transfer of total Cd is another basis for the TF factor (Takarina and Pin, 2017). 3.4.3. Health Risk Assessment for Grazing Animals As a general trend, in treatments T1-T4, the value of PLI i.e., the level of metal contamination was consistently zero, indicating no detectable pollution load. HRI, DIM, and PLI from T1 to T4 can be declared as 'no-risk', 'safe' or 'low-risk', hence posing no adverse health effects to the grazing animals (Table 1 ). However, the highest PLI was observed in treatment T5 (0.037), followed by T7 (0.015), T6 (0.013), and T8 (0.009), suggesting that these treatments were associated with varying levels of metal Contamination. The highest Cd contamination in T5 calls for concern, particularly among people who consume animal products. In terms of metal intake, cows, sheep, and buffalo showed no daily intake of metal for treatments T1-T4, indicating the absence of metal exposure in these treatments (Table 2). Treatment T5 exhibited the highest DIM values for all three species (0.003 for cows, sheep, and buffalo), indicating significant metal ingestion. The metal intake in treatments T7 and T8 was negligible, as indicated by very low DIM values (0.000). Likewise, highest HRI was observed with T5 (0.221 for cows, 0.192 for sheep, and 0.252 for buffalo), suggesting the greatest health risk, especially for buffalo. The low HRI values observed in treatments T7 and T8 (T7: 0.004 for cows, 0.003 for sheep, 0.002 for buffalo; T8: 0.006 for cows, 0.005 for sheep, 0.006 for buffalo) indicated minimal health risks. 3.5. Principle Component Analysis (PCA) Physiological and biochemical parameters of maize were analyzed by constructing biplots of PCA to evaluate the most efficient treatment in study. PCAs contributed to 94.34% variance in plant biplots (Fig. 7 ). Following that, proteins had a strong positive relationship with CAT, APX, SOD, and POD but a strong negative relationship with H2O2 and MDA (Fig. 7 ). Similarly, Cd showed a highly negative correlation with proteins, CAT, phenolics, APX, SOD, and POD, while Cd showed positive correlation with H2O2 and MDA. The PCA results showed that addition of Si and SN215 improved antioxidant enzymes and secondary metabolites in maize plants. The PCA results significantly investigated that the application of T8 has the greatest effect on growth and biochemical parameters of maize crops under cadmium stress. 4. Discussion 4.1 Biosorption Trial on SN215 In our study, biosorption trial with cadmium concentrations of 5 ppm (low), 10 ppm (medium), and 15 ppm (high), biosorption efficiency was calculated after 24 hours and 48 hours, it was shown that biosorption effectiveness peaked at 10 ppm. (Fig. 2 ). The result fits with what we know about how the concentration of heavy metals in a solution can affect biosorption processes. This is because higher concentrations may make binding sites on the surface of bacteria cells full, which makes biosorption less effective at high levels. A study found that extending the contact period beyond 24 hours reduced the effect size and removal efficiency for Cd (II). The increased toxicity of the metal ions to living bacterial strains may cause a decrease in biosorption capacity with longer contact time (Ibuot et al., 2017; Igiri et al., 2018 ). Contact lengths longer than 24 hours can impact the metal speciation, solubility, and eventual desorption or precipitation of metal ions, while the metabolic activities of living bacteria can cause pH variations in the metal solution (Ratzke et al., 2018; Xie et al., 2018). At 10 ppm, the biosorption efficiency remained steady, indicating that this dosage is optimum for the bacterial strains studied. The abundance of binding sites and the maintenance of the bacteria's metabolic activity without the inhibitory effects observed at higher concentrations may be responsible for this stability (Hossain and Aditya, 2015). The results show that the bacterial strains used in the biosorption studies are very good at absorbing cadmium at low to moderate levels. This is important for making bioremediation methods that work well (Fig. 2 ). 4.2 Effect of SN215 on Maize Fodder Growth and Yield The growth of maize fodder was significantly reduced because of phytotoxic nature of Cd, especially affecting the shoot fresh and dry weights (Fig. 2 ). Biomass serves as a basic ecological parameter to determine plant growth and reflects the plants' ability to manage nutritional reservoirs and provide necessary data to evaluate the plant's potential under stressful conditions (Cid et al., 2020 ). Cadmium aggregation reduces seed germination, primary seedling growth, and overall plant biomass. It alters the rate of photosynthesis, transpiration rate, relative water content, electrolyte leakage and stomatal conductance. Cd stress switches on the production of ROS, which leads to chromosomal abnormalities, genetic mutations, and DNA impairment, hence affecting the cell division as well as cell cycle (El Rasafi et al., 2022 ). Shah et al. have demonstrated that Cd stress significantly reduced fresh as well as dry biomass in maize (Shah et al., 2023 ). Our results demonstrate that application of Si alone or in combination with Rhizobacteria is a substantial mitigation of Cd stress, with a two-fold increase in shoot fresh weight when Si was applied along with Rhizobacteria. Si likely improved plant tolerance by strengthening cell walls, while Rhizobacteria may have enhanced nutrient uptake or immobilized Cd in the soil. These results suggest that combining Si and Rhizobacteria can be an effective strategy for improving crop growth under heavy metal stress. 4.3 Effect of Si and SN215 on Maize Fodder Quality One of the many reasons for Cd-triggered growth inhibition may be associated with decreased relative water content in plants stressed with Cd, as reported previously (Kaya et al., 2020 ). In this experiment, the combined application of Si and Rhizobacteria significantly improved the RWC of Cd-stressed plants, indicating that the treatment helped maize fodder plants maintain turgor pressure even in the presence of Cd toxicity. Our results are analogous to those of Azizi et al, where improvement in relative water content of savory plants improved plant biomass under Cd stress (Azizi et al., 2020 ). Heavy metals hinder photosynthesis, affecting the generation of chlorophyll and carotenes. Moreover, HMs stress affects the activities of key enzymes involved in photosynthesis. Most importantly, HM stress causes the down-regulation of light-harvesting compounds chlorophyll and chlorophyll b, restricting photosynthesis and ultimately affecting plant development (Zhou et al., 2024 ). Likewise, in the present study, the concentration of chlorophyll content (chlorophyll a and b) was reduced significantly with applied stress under T4 (10ppm Cd) as compared to control T1. However, the combined application of Si and SN215 significantly improved photosynthetic pigments (chlorophyll a, chlorophyll b and xanthophyll) of maize plants. The synergistic effect of Si and SN215 probably enhanced the photosynthetic pigments in maize by improving nutrient uptake, strengthening the plant structure, protecting plants against oxidative stress, and stimulating growth-promoting processes. This synergistic effect resulted in better overall plant health and higher photosynthetic efficiency. The improved nutrient uptake, water status, and hormonal balance in the plant could be the cause of the increased photosynthesis rate. Our results are in parallel with studies carried out by Cipriano et al., 2021 ; Kollárová et al., 2019 and Sarathambal et al., 2017 in which structure of chloroplast was improved using Si and SN215 under Cd stress. Proteins and phenolic compounds play an important role in maintaining proper structure and function of plant cells. All the important biochemical reactions are liked with proper functioning of protein and phenolics (Goncharuk et al., 2023). According to previous reports, Cd has the potential to reduce the overall protein content of plants by restricting the uptake of potassium and magnesium, promoting post-translational modifications, reducing the rate of photosynthesis, enhancing protein degradation, and prohibiting RuBisCo activity (Monteiro et al., 2009 ). Under Cd stress, the concentration of phenolic compounds, known as metal-chelating agents, increases (Goncharuk and Zagoskina, 2023 ). However, the presence of Cd stress significantly reduced the concentration of proteins and phenolics (Fig. 4 ), potentially due to Cd-generated toxicity. The application of Si and Lysinibaciluus sp. significantly improved the concentration of proteins and phenolics in T7 compared to Cd stress (T4) alone (Fig. 4 ). Similar results have been reported in previous studies where individual applications of Si and SN215 have helped HM-stressed plants combat heavy metal stress by increasing total protein and phenolic content (Mihaličová Malčovská et al., 2014 ; Anwaar et al., 2015 ; Zheng et al., 2023 ). 4.4 Effect of Si and SN215 on Cd Tolerance Capacity of Maize Fodder Heavy metal stress disturbs the dynamic equilibrium of ROS production in plants. These ROS start accumulating in membranes of plants, which disturbs structure and functions of cell membranes. It has been documented that high level of Cd can cause production of malondialdehyde (MDA) and hydrogen peroxide (H 2 O 2 ), which are indicators of cell membrane damage (Deng et al., 2024). Our research showed that Cd stress made oxidative damage much worse in maize plants. This was shown by high levels of MDA and H2O2, which are signs of lipid peroxidation and oxidative stress. A one-fold increase in MDA and a five-fold increase in H 2 O 2 was observed under 10 ppm Cd stress. However, the application of Siand SN215 effectively mitigated oxidative damage, reducing MDA by 61.96% and H 2 O 2 by 59.43%. There are two ways by which Cd-generated oxidative stress burden can be mitigated: enzymatic and non-enzymatic antioxidant defence systems, which can maintain redox homeostasis. The enzymatic defence antioxidants enzymes such as ascorbate peroxidase (APX), catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), and dehydroascorbate reductase (DHAR). The non-enzymatic pathway scavenges ROS with the help of redox or antioxidant metabolites, such as glutathione (GSH), ascorbate (AsA), nicotinamide adenine dinucleotide phosphate (NADPH) to lighten the oxidative stress (Jung et al., 2020). In the current study, cadmium stress (10 ppm) significantly inhibited the activities of key antioxidant enzymes, including APX, CAT, SOD, and POD in maize. This reduction in enzyme activity reflects the disruption of the plant's ability to mitigate ROS generated under Cd stress. The big drop in shoot APX (88.2%) and CAT (67%) suggests that oxidative stress caused by Cd makes it much harder for cells to get rid of hydrogen peroxide and other ROS, which can damage cells. The interactive role of Si and PGPBs may alleviate the oxidative damage in numerous plant species via upregulation of ROS-scavenging enzymes along with non-enzymatic branch of antioxidation (Kim et al., 2017 ; Verma et al., 2020 ). Interestingly, in our current study, Cd-stressed plants, when inoculated with SN215 as well as supplied with Si, the activities of antioxidant enzymes were significantly increased compared to the control (Fig. 3 ), highlighting the potential of Si and SN215 to bolster the antioxidant defense system in maize under Cd stress. Our results are clearly in line with those of Farooq et al. where application of Si effectively mitigated the deleterious effects of Cd stress in cotton by decreased production of MDA and H 2 O 2 hence reducing net oxidative stress burden on the plants (Farooq et al., 2013 ). Our results are also in parallel with studies of Luyckx et al., 2021 ; Das et al., 2021 in which growth of Cannabis sativa L. and rice ( Japonica and Indica) was improved by decreasing the concentration of ROS using Si and SN215 under heavy metal stress. 4.5 Effect of Si and SN215 on Cd Contents in Soil and Shoots of Maize Fodder Cd is extremely mobile once inside the plant tissues and greatly impacts physiological growth (Saidi et al., 2013 ). In the current study, the concentration of Cd significantly increased with applied stress (10ppm Cd) compared to control (Fig. 5 ). However, the application of Si and SN215 proved to be beneficial in reduction of bioavailability of heavy metals to plants by different possible mechanisms. Si causes immobilization of heavy metals (Sahebi et al., 2015 ). Soil pH plays an important role in immobilization of heavy metals by formation of silicate complexes. Heavy metals contaminated soils, which are amended by Si + Cd is absorbed by oxides of iron and manganese. So, bioavailability of heavy metals can be limited to plants due to formation of silicate complexes (Ma et al., 2021 ). Photosynthesis is a crucial physiological process for plants, and as per numerous reports, this process is severely inhibited by Cd stress. The rate of photosynthesis inhibition is directly proportional to the concentration of Cd (Küpper et al., 1996 ; Zhao et al., 2021 ). Consistent with previous studies, the application of Cd caused a drastic reduction in the chlorophyll a and b content (Fig. 4 C). The reduction in chlorophyll content was possible because of inhibition of synthesis of chlorophyl due to Cd toxicity. However, the combined application of Si and SN215 significantly improved photosynthetic pigments of maize plants (Fig. 4 C). The observations made in the current study are in accordance with previous reports where application of Si minimized the damage to chlorophyll content in Zea mays plants under Cd toxicity (Saleem et al., 2022 ) and application of plant growth promoting bacteria ( Pseudomonas aeruginosa and Burkholderia gladioli ) in Lycopersicon. esculentum seedlings improved chlorophyll content of seedlings under Cd stress as compared to control ones (Khanna et al., 2019 ). The increased under Cd stress chlorophyll content in our study after application of Si and SN215 is perhaps due to Cd chelation by Si and SN215, hence restricted uptake of Cd in plants. Moreover, inoculation of plants with bacteria might have enhanced the nutrient uptake, improving photosynthetic potential of plants under Cd toxicity. 4.6 Effect of Si and SN215 on Health Risk Assessment for Grazing Animals Estimation of exposure level of heavy metals, along with tracking their route of contamination for target organisms, is crucial for comprehension of associated health risks (Li et al., 2014 ). Health risks linked with atmospheric contamination possibly arise from the inhalation of heavy metal particles or from consuming polluted food (Xiong et al., 2014 ; Shahid et al., 2017 ). The pollution load index (PLI) results for Cd metal ranged from 0.427–0.805 in the soil samples (Table 2). T8 practised the minimum value, while T5 represented the maximum value. Overall, the values of pollution load index were observed to be less than 1, which indicated that all the treatments are unpolluted (Khan et al., 2019 ). The values reported by Ezemokwe et al. ( 2017 ) and Khan et al. ( 2020 ) were 0.05 and 0.0649 mg/kg, which are observed to be lower than the observed concentration in our studies. The observed PLI range was less than 1 in all treatments, which represents that all treatments reduced Cd toxicity in present research. DIM (daily intake of metal) presents an index for determination of body exposure to heavy metal through feeding (Daniele et al., 2019 ). The daily Cd intake varied from 0.00 to 0.004 mg/kg/day. The study focused on the minimum intake in sheep, while the buffaloes grazing on Zea mays perceive the maximum concentration. The HRI value for Cd amounted to 0.00 mg/kg/day and was 0.252 mg/kg/ day. The maximum value of HRI was analysed in the buffaloes, while the minimum level was assessed in the sheep (Table 2). Similar results were observed by Khan et al. ( 2020 ), who conducted a pot trial to assess the impact of wastewater on zinc accumulation in various forages and their associated health risks. They grew both summer forages (e.g., Zea mays, Sorghum bicolor) and winter forages (e.g., Trifolium alexandrinum, Brassica napus) using sewage and tap water treatments. Results showed that the pollution load index, daily intake of metal, and health risk index for zinc were all below 1, indicating that the consumption of these forages posed no health risk. This study showed the DIM range (0.00164– 0.0813mg/kg/day), which is like present results. 5. Conclusions Silica with SN215 showed efficient performance in reducing cadmium toxicity in Zea mays crop through scavenging burst production of ROS, stimulating the enzymatic and non-enzymatic defence mechanisms, and improving photosynthetic machinery of experimental plants. Hence application of silica in combination with SN215 has proved to be an eco-friendly soil conditioner strategy that can be used in developing countries to limit the deleterious effects of Cd on fodder crops. Overall, the data indicated that treatment T5 poses the most significant health concerns due to its higher pollution load, metal intake, and associated health risks, particularly for buffalo. Treatment T6 also posed a moderate risk, while treatments T7 and T8 showed only minimal impact. Declarations CRediT authorship contribution statement Seemal Naeem: Writing – review & editing, Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Conceptualization. Waqas ud Din Khan: Formal analysis, Conceptualization, Investigation, Resources, Project administration. Tahmina Nazish: Writing – review & editing, Supervision, Methodology, Funding acquisition, Formal analysis, Conceptualization. Muhammad Umer Farooq Awan: Formal analysis, Writing – review & editing, Resources. Usman Ijaz: Writing – review & edit- ing, Supervision, Methodology, Funding acquisition, Formal analysis, Conceptualization. Abdul Sattar Nizami: Writing – review & edit- ing, Supervision, Funding acquisition, Formal analysis. Mohsin Tanveer: Writing – review & editing, Supervision, Funding acquisition. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data availability Data will be made available on request. References Ahmad P, Abdel Latef AA, Abd_Allah EF, Hashem A, Sarwat M, Anjum NA, Gucel S (2016) Calcium and potassium supplementation enhanced growth, osmolyte secondary metabolite production, and enzymatic antioxidant machinery in cadmium-exposed chickpea (Cicer arietinum L). 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BioMed research international, 2015(1), 396010 Saidi I, Ayouni M, Dhieb A, Chtourou Y, Chaïbi W, Djebali W (2013) Oxidative damages induced by short-term exposure to cadmium in bean plants: protective role of salicylic acid. South Afr J Bot 85:32–38. https://doi.org/10.1016/j.sajb.2012.12.002 Saleem MH, Parveen A, Khan SU, Hussain I, Wang X, Alshaya H, El-Sheikh MA, Ali S (2022) Silicon fertigation regimes attenuates cadmium toxicity and phytoremediation potential in two maize (Zea mays L.) cultivars by minimizing its uptake and oxidative stress. Sustainability 14:1462. https://doi.org/10.3390/su14031462 Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual (No. Ed. 2, pp. xxxviii+-1546) Sapunar-Postružnik J, Bažulić D, Grubelić M, Drinčić K, H., and, Njari B (2001) Cadmium in animal feed and in foodstuffs of animal origin. 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Plant Cell Rep, 1–13 U.S. Environmental Protection Agency (2020) Human health risk assessment Verma KK, Song X-P, Li D-M, Singh M, Rajput VD, Malviya MK, Minkina T, Singh RK, Singh P, Li Y-R (2020) Interactive role of silicon and plant–rhizobacteria mitigating abiotic stresses: A new approach for sustainable agriculture and climate change. Plants 9:1055. https://doi.org/10.3390/plants9091055 Walkley A (1947) A critical examination of a rapid method for determining organic carbon in soils—effect of variations in digestion conditions and of inorganic soil constituents. Soil Sci 63(4):251–264 Weisberg RA, Gottesman ME, Hendrix RW, Little JW (1999) Family values in the age of genomics: comparative analyses of temperate bacteriophage HK022. Annu Rev Genet 33(1):565–602 Wen J, Dong H, Zeng G (2018) application of zeolite in removing salinity/sodicity from wastewater: A review of mechanisms, challenges and opportunities. 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J Soil Sci Plant Nutr 20:1110–1121. http://dx.doi.org/10.1007/s42729-020-00197-y Yan G, Jin H, Yin C, Hua Y, Huang Q, Zhou G, Xu Y, He Y, Liang Y, Zhu Z (2023) Comparative effects of silicon and silicon nanoparticles on the antioxidant system and cadmium uptake in tomato under cadmium stress. Sci Total Environ 904:166819. http://dx.doi.org/10.1016/j.scitotenv.2023.166819 Yerli C, Sahin U, Oztas T, Ors S (2024) Fertility and heavy metal pollution in silage maize soil irrigated with different levels of recycled wastewater under conventional and no-tillage practices. Irrig Sci 1–18. http://dx.doi.org/10.1007/s00271-024-00927-5 Zafar-Ul-Hye M, Shahjahan A, Danish S, Abid M, Qayyum MF (2018) Mitigation of cadmium toxicity induced stress in wheat by ACC-deaminase containing PGPR isolated from cadmium polluted wheat rhizosphere. Pak J Bot 50(5):1727–1734 Zhao H, Guan J, Liang Q, Zhang X, Hu H, Zhang J (2021) Effects of cadmium stress on growth and physiological characteristics of sassafras seedlings. Sci Rep 11(1):9913 Zheng Y, Tang J, Liu C, Liu X, Luo Z, Zou D, Xiang G, Bai J, Meng G, Liu X (2023) Alleviation of metal stress in rape seedlings (Brassica napus L.) using the antimony-resistant plant growth-promoting rhizobacteria Cupriavidus sp. S-8-2. Sci Total Environ 858:159955. https://doi.org/10.1016/j.scitotenv.2022.159955 Zhou R, Xu J, Li L, Yin Y, Xue B, Li J, Sun F (2024) Exploration of the Effects of Cadmium Stress on Photosynthesis in Oenanthe javanica (Blume) DC. Toxics 12, 307. https://doi.org/10.3390/toxics12050307 Zhu T, Li L, Duan Q, Liu X, Chen M (2021) Progress in our understanding of plant responses to the stress of heavy metal cadmium. Plant Signal Behav 16(1):1836884. http://dx.doi.org/10.1016/j.jhazmat.2020.123608 Supplementary Files GRAPHICALABSTRACT.png SupplementaryFigandTables2642025.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6592002","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":453013672,"identity":"b1f5c742-7d4b-4269-a359-258ad94ff235","order_by":0,"name":"Seemal Naeem","email":"","orcid":"","institution":"Government College University Lahore","correspondingAuthor":false,"prefix":"","firstName":"Seemal","middleName":"","lastName":"Naeem","suffix":""},{"id":453013673,"identity":"63c5de56-0bdc-461e-8d0e-02a861ee0193","order_by":1,"name":"Waqas khan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAUlEQVRIiWNgGAWjYDCCA3BWgsEBhgoIUwKIDYjUcoZULQyMbURo4bt9gE3iR5mdvcHx5I2HC+fVyhscYD54m4fhjjEuLZLnEtgke84lJ24486zg8Mxtxw03HGBLtuZheGaGS4vBGQa2G7xtzAkGN3IMDvNuOwb0EY+ZNA/DYRt8Wm7+bau3h2iZA9LC/42gltu8bYcZN4C1NNSAbGEDacHpMMkzjO2/Zc4dT5wJ8gvPsQOGMw+zGVvOMXiG0/t8Z5gPG74pq7bnO568+TNPTZ083/HmhzfeVNwxbMClh4ERKMUG5x1mYGAGO/gATg0QgNBSB2MQ0jIKRsEoGAUjCAAAXxld8RiuB/8AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-6704-3887","institution":"Government College University Lahore","correspondingAuthor":true,"prefix":"","firstName":"Waqas","middleName":"","lastName":"khan","suffix":""},{"id":453013674,"identity":"a6480838-9ae5-484f-9f08-58036fd0d0c0","order_by":2,"name":"Tahmina Nazish","email":"","orcid":"","institution":"University of Western Australia Faculty of Natural and Agricultural Sciences: The University of Western Australia Faculty of Science","correspondingAuthor":false,"prefix":"","firstName":"Tahmina","middleName":"","lastName":"Nazish","suffix":""},{"id":453013675,"identity":"1e0e37bb-183e-450e-b9bb-f1225c4b9787","order_by":3,"name":"Muhammad Awan","email":"","orcid":"","institution":"Government College University Lahore","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"","lastName":"Awan","suffix":""},{"id":453013676,"identity":"edd8a72f-ac78-4479-bc75-03c784f66806","order_by":4,"name":"Usman Ijaz","email":"","orcid":"","institution":"University of Tasmania - Inveresk Campus: University of Tasmania - Launceston Campus","correspondingAuthor":false,"prefix":"","firstName":"Usman","middleName":"","lastName":"Ijaz","suffix":""},{"id":453013677,"identity":"e35edb62-baeb-4fca-88d9-e6792df38458","order_by":5,"name":"Abdul-Sattar Nizami","email":"","orcid":"","institution":"Government College University Lahore","correspondingAuthor":false,"prefix":"","firstName":"Abdul-Sattar","middleName":"","lastName":"Nizami","suffix":""},{"id":453013678,"identity":"7c7ca626-e779-4c34-a967-24427d3b2bec","order_by":6,"name":"Mohsin Tanveer","email":"","orcid":"","institution":"Xinjiang Institute of Ecology and Geography","correspondingAuthor":false,"prefix":"","firstName":"Mohsin","middleName":"","lastName":"Tanveer","suffix":""}],"badges":[],"createdAt":"2025-05-05 07:13:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6592002/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6592002/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82606983,"identity":"412edb4f-8ba7-4ed6-a8cd-e54abf329576","added_by":"auto","created_at":"2025-05-13 10:10:06","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":907482,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification and phylogenetic analysis of SN215: (A) Growth of SN215 on nutrient agar media (B) DNA bands after polymerase chain reaction (PCR), DNA marker = 1kb\u003cstrong\u003e (C)\u003c/strong\u003ePhylogenetic analysis of query isolate (SN215) by using maximum likelihood method on the bases of Kimura 2-parameter model (in MEGA X)\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6592002/v1/a80dc7360c5cda46f5d5aae2.jpeg"},{"id":82603844,"identity":"4f2cfe88-d027-4824-90d9-ff34eca79c84","added_by":"auto","created_at":"2025-05-13 09:54:06","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":245051,"visible":true,"origin":"","legend":"\u003cp\u003eFresh weight (FW in grams) and dry weight (DW in grams) in shoot of maize plants was determined under different treatments against cadmium stress such as T1 = Control; T2 = 2mM Silicon; T3 =Bacteria; T4 =2mM Silicon + Bacteria; T5= Cd (Medium level); T6 = 2mM Silicon + Cd (Medium level); T7 = Bacteria + Cd (Medium level); T8 = 2mM Silicon + Bacteria + Cd (Medium level). Values are the average of three replicates ± standard error (SE).\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6592002/v1/f8cb889aa1e84324ea39ec25.jpeg"},{"id":82605508,"identity":"ebfef83a-275e-4723-be9f-5653fa66870c","added_by":"auto","created_at":"2025-05-13 10:02:06","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":241642,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of application of silicon and SN215 on growth and biochemical parameters of maize: \u003cstrong\u003e(A)\u003c/strong\u003e Relative water content (%), \u003cstrong\u003e(B)\u003c/strong\u003e Chlorophyll content (mg ml\u003csup\u003e-1\u003c/sup\u003e), \u003cstrong\u003e(C)\u003c/strong\u003e Total phenolics (µg g-\u003csup\u003e1\u003c/sup\u003e), and\u0026nbsp; \u003cstrong\u003e(D)\u003c/strong\u003e Total protein (µg g-\u003csup\u003e1\u003c/sup\u003e) concentration in shoot of\u0026nbsp; maize plant were determined under different treatments against cadmium stress such as T1 = Control; T2 = 2mM Silicon; T3 =Bacteria; T4 =2mM Silicon + Bacteria; T5= 10 ppm Cd; T6 = 2mM Silicon + 10 ppm Cd; T7 = Bacteria + 10 ppm Cd; T8 = 2mM Silicon + Bacteria + 10 ppm Cd. Values are the average of three replicates ± standard error (SE).\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6592002/v1/391635d309fe61e634dc52f5.jpeg"},{"id":82605505,"identity":"4967bcf2-7184-47d0-a97e-2e4cdef68c87","added_by":"auto","created_at":"2025-05-13 10:02:06","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":579322,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of silicon and bacterial inoculation on \u003cstrong\u003e(A)\u003c/strong\u003e Malondialdehyde (nmol ml\u003csup\u003e−1\u003c/sup\u003e) and hydrogen peroxide (µmol g\u003csup\u003e-1\u003c/sup\u003e )\u003cstrong\u003e (B)\u003c/strong\u003e Ascorbate peroxidase (µmol g\u003csup\u003e−1\u003c/sup\u003e) and catalase (µmol g\u003csup\u003e−1\u003c/sup\u003e), and\u0026nbsp; \u003cstrong\u003e(C) \u003c/strong\u003eSuper Oxide Dismutase (mg protein\u003csup\u003e-1\u003c/sup\u003e)\u003cstrong\u003e \u003c/strong\u003eand peroxidase (unit mg protein\u003csup\u003e-1\u003c/sup\u003e) in shoots of\u0026nbsp; maize plant were determined under different treatments against cadmium stress such as: T1 = Control; T2 = 2mM Silicon; T3 =Bacteria; T4 =2mM Silicon + Bacteria; T5= 10 ppm Cd; T6 = 2mM Silicon + 10 ppm Cd; T7 = Bacteria + 10 ppm Cd; T8 = 2mM Silicon + Bacteria + 10 ppm Cd. Values are the average of three replicates ± standard error (SE).\u003c/p\u003e","description":"","filename":"floatimage4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6592002/v1/3564af3db51196403daadf01.jpg"},{"id":82606984,"identity":"7e198ff4-f112-4e5d-8914-8804d489cda3","added_by":"auto","created_at":"2025-05-13 10:10:06","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":278823,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of application of silicon and SN215 on cadmium uptake in maize and soil: \u003cstrong\u003e(A)\u003c/strong\u003e Cd uptake in shoot (mg g\u003csup\u003e-1\u003c/sup\u003e DW) \u003cstrong\u003e(B)\u003c/strong\u003e soil Cd concentration (mg kg\u003csup\u003e-1\u003c/sup\u003e) (C) Bioconcentration Factor (BCF) (D) Translocation Factor (TF) under different treatments against cadmium stress such as T1 = Control; T2 = 2mM Silicon; T3 =Bacteria; T4 =2mM Silicon + Bacteria; T5= Cd (Medium level); T6 = 2mM Silicon + 10 ppm Cd; T7 = Bacteria + 10 ppm Cd; T8 = 2mM Silicon + Bacteria + 10 ppm Cd. Values are the average of three replicates ± standard error (SE).\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6592002/v1/2980485454f8fd988a7b9ba4.jpeg"},{"id":82603853,"identity":"955179b0-76c5-4e7a-9422-1d8202920bfe","added_by":"auto","created_at":"2025-05-13 09:54:06","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":429217,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal component analysis (PCA) of maize plants and soil. F1 and F2 are PCA main factors which significantly contributed to generate PCA biplots. Active variables are fresh weight, dry weight, chlorophyll a (Chl. a), Chlorophyll b (Chl. b), hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), malondialdehyde (MDA), ascorbate peroxidase (APX), catalase (CAT), proteins and phenolics. Active observants are silicon and Lysinibacillus sp.\u003c/p\u003e","description":"","filename":"floatimage6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6592002/v1/a9019d993c8a646c65a1dfcb.jpg"},{"id":82748017,"identity":"aedb02af-b4ac-4d1d-97a8-d0d9d79c4216","added_by":"auto","created_at":"2025-05-14 19:28:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3945989,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6592002/v1/3875b233-8e8e-4204-9cbd-146ba27a98d5.pdf"},{"id":82605506,"identity":"9002fd9d-e0df-4d99-a940-536ea488ac06","added_by":"auto","created_at":"2025-05-13 10:02:06","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":679530,"visible":true,"origin":"","legend":"","description":"","filename":"GRAPHICALABSTRACT.png","url":"https://assets-eu.researchsquare.com/files/rs-6592002/v1/eeb23b83c2afcfcc4f26d9e0.png"},{"id":82603841,"identity":"6c8853bf-f1d7-4249-8f34-cd6a9af1138a","added_by":"auto","created_at":"2025-05-13 09:54:06","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":14497,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigandTables2642025.docx","url":"https://assets-eu.researchsquare.com/files/rs-6592002/v1/92b163a7fabd3ea95add5ea4.docx"}],"financialInterests":"","formattedTitle":"Unravelling Potential of Silicon and Rhizobacteria in Reducing Cd-related Health Risk in Grazing Animals by Enhancing Maize Fodder Quality","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eWastewater application in the agriculture sector of developing countries has been gaining attention in recent years due to freshwater scarcity (FAO, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Yerli et al., \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Wastewater or industrial effluents contain toxic and heavy metal ions (even after recycling), which ultimately deteriorate the fodder yield and quality (Wen et al., \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Islam et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Heavy metals in wastewater slow plant growth by creating reactive oxygen species (ROS) or by changing the functions of important enzymes by taking away metals and nutrients that plants need (Zhu et al., \u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Alsafran et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Hu et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). For instance, Cadmium (Cd) enrichment in several soils across the globe was linked with the use of Cd rich industry effluent or wastewater for irrigation purposes (Rezapour et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The Cd ranked seventh among potential hazardous elements with significant health and environmental health related hazards (Genchi et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCd enriched fodder crops showed reduced fodder quality, such as reduced growth and increased electrolyte leakage and enhanced production of ROS in \u003cem\u003eSorghum bicolor\u003c/em\u003e (Hassan et al., 2020), reduced plant height as well as biomass in \u003cem\u003eMedicago sativa\u003c/em\u003e (Kareem et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), reduced shoot and root length, dry and fresh weight, and chlorophyll content in \u003cem\u003eZea mays\u003c/em\u003e (Shah et al., \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), decreased protein as well as lipid content in \u003cem\u003eOryza sativa\u003c/em\u003e (Lan et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and amount of carbohydrates and their conjugates in \u003cem\u003ePanax notoginseng\u003c/em\u003e (Lu et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Studies have shown that several animals' consumption of Cd-rich fodder is a major source of Cd toxicity (Sapunar-Postrunik et al., 2001). A significant accumulation of Cd has been reported in faeces, hair, and blood samples of cows and buffalos fed with Cd-contaminated forage crops. Residents exposed to the milk and meat of these cattle showed serious health concerns (Hussain et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Maize, being one of most important cereal crops, is susceptible to cadmium stress and maize products safety is directly linked to food safety as Cd can be transferred from fodder to animals and then to humans, hence being a potential threat to both animals and humans (Liu et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Lamb et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Rizwan et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe accumulation of Cd at high concentrations in maize tissues reduces shoot growth, plant height, plant fresh and dry weight, activity of antioxidant enzymes, chlorophyll content and ultimately, poor fodder quality (He et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Ruiz-Huerta et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). It was found that Cd decreased the yield of fodder crops by stopping roots from growing longer, messing up nutrient uptake, stopping reproductive development, slowing down photosynthesis, and ultimately lowering yield in higher plants (Anjum et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). At the cellular level, Cd induces oxidative stress, destroying the cellular structures which leads to cellular death (Clemens et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Shanying et al., \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMoreover, Cd induced greater oxidative damage and accumulation of malondialdehyde (MDA) concentration in the shoot and leaf of fodder crops was associated with a decline in fodder quality (Ahmad et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Pandian et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Awan et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This prompts an examination of methods for reducing Cd toxicity in fodder crops, particularly maize fodder. Examining approaches to reduce Cd uptake and its translocation from root to shoot could provide the answer, leading to an improvement in maize fodder yield and quality. One can argue that developing tolerant crop varieties using gene editing techniques could be a more productive solution. However, the use of gene editing techniques is greatly influenced by expert personnel, plant species, and experimental conditions, thus indicating the adoption of agronomic techniques as a green technology for ameliorating Cd toxicity in fodder crops.\u003c/p\u003e \u003cp\u003eTraditional physicochemical methods using hydrated lime for neutralization of soil Cd and soil replacement for Cd remediation are highly effective, but their applications are limited due to high costs and soil pollution (Lim et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Bian et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Kumpiene et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Mu et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2024\u003c/span\u003e)). Therefore, there is an urgent need to develop innovative techniques to sustain maize fodder production and mitigate Cd toxic effects. When it comes to cleaning up metal-contaminated farmland, biological methods using different types of plant growth-promoting rhizobacteria (PGPR) have gotten much attention (Zafar-Ul-Hye et al., \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Khanna et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Ahmed et al., 2024). PGPRs are very good at keeping heavy metals in place and stopping them from moving around in plants by forming complexes, precipitating them, and absorbing them (Khanna et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Researchers have extensively studied the PGPR's inherent metal immobilization capabilities and metal resistance to promote plant growth and reduce metal accumulation in plants. Moreover, metal-resistant bacteria release growth-promoting substances in plants' rhizosphere, such as solubilized phosphates, siderophores, and indole-3-acetic acid, to confer tolerance in plants under a metal-contaminated environment (Kumar et al., 2023; Ijaz et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Therefore, this study examined the role of SN215 in reducing Cd toxicity in maize fodder.\u003c/p\u003e \u003cp\u003eShoot development is one of the most important plant traits in fodder crops, as higher shoot growth and development produce higher fodder yield with better fodder quality traits (Prajapati et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In this context, Salicilic acid (Si) is important for its critical role in improving shoot growth, rigidity, and stress tolerance (Mir et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ali and Bijay, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The Si application improved shoot growth by improving nutrient uptake, which later improved shoot length, shoot fresh weight, shoot relative contents and shoot greenness (higher chlorophyll contents) (Akhter et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) Moreover, the beneficial role of Si in conferring Cd tolerance was associated with (i) protecting ROS induced cell death (Xuebin et al. \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), (ii) higher chlorophyll contents (Yan et al., \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and (iii) higher cellular turgidity and elasticity (Jan et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe application of Si was also examined for conferring Cd toxicity in maize fodder. Moreover, the combined application of Si was examined for improving maize fodder yield and Cd related health risks in grazing animals. Hence, a study was conducted to evaluate the combined role of Si and \u003cem\u003eLysinibacillus\u003c/em\u003e sp. in mitigating the Cd toxicity in maize fodder. Furthermore, we tend (i) to examine the synergistic impact of Si and \u003cem\u003eLysinibacillus\u003c/em\u003e sp. in improving growth parameters and fodder quality traits, such as chlorophyll content, protein levels, and water retention under Cd stress (ii) the effectiveness of Si and \u003cem\u003eLysinibacillus\u003c/em\u003e sp. in reducing oxidative stress markers (e.g., MDA, H₂O₂) and enhancing the antioxidant defense system in maize fodder under Cd stress; lastly, (iii) to analyze the potential of Si and \u003cem\u003eLysinibacillus\u003c/em\u003e sp. to limit Cd uptake/ its bioavailability in maize fodder shoots and assess the health risk index (HRI) for grazing animals consuming Cd-contaminated fodder.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Isolation and Identification of Rhizobacteria\u003c/h2\u003e \u003cp\u003eSoil samples were collected from the rhizosphere of wheat crops in South Punjab, Pakistan, and stored in plastic bags at 4\u0026deg;C until further analysis. Serial dilutions (10⁻\u0026sup1; to 10⁻⁹) were prepared by taking 10 g of rhizosphere soil. Microorganisms from each dilution were then cultured on petri plates, which were incubated at 37\u0026deg;C for 24 hours. After incubation, colonies of varying sizes and shapes were observed on the media, representing mixed cultures. To isolate pure colonies, each colony was streaked onto separate petri plates. Repeated streaking was performed to purify the bacterial isolates (Loutfi et al., 2020). These purified cultures were incubated again at 37\u0026deg;C for 24 hours. DNA was extracted from the 24-hour-old cultures following the optimized method of Sambrook et al. (\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e1989\u003c/span\u003e) and stored in double-distilled deionized water at -20\u0026deg;C for further analysis. The 16S rRNA gene was amplified using a set of universal primers, fD1 and rD1, as previously described (Weisburg et al., 1999). The samples were sent to company for sequencing 16s RNA gene. ClustalW was used to perform multiple sequence alignment of the 16S rRNA gene sequence with its homologous sequences. The phylogenetic tree was constructed using MEGA (v.11.0) software, using the Maximum Likelihood (ML) method with 1000 bootstrap replicates to measure evolutionary relationships.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Cd biosorption and Characterization of Bacterial Isolate\u003c/h2\u003e \u003cp\u003eGram staining was performed to determine the bacterial characteristics. Morphological characterization, including the examination of cell shape and colony structure, was conducted using a compound microscope (model XSZ107BN-A11.1007-17, China). The biosorption of total Cd was investigated by inoculating pure bacterial isolate W2 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) into broth media. To assess the efficiency of SN215 in capturing cadmium (Cd), three different levels of Cd were used: low (5 ppm), medium (10 ppm), and high (15 ppm). A 1 mL volume of overnight bacterial cultures grown on petri plates was introduced into 100 mL of sterilized broth using 15 mL falcon tubes. The tubes were incubated at 30\u0026deg;C for 24 hours. After incubation, the bacterial cultures were centrifuged at 13,000 revolutions per minute (rpm) for 10 minutes. The supernatants were collected to measure the remaining Cd content using a multi-sequential AAS (iCE 3000 SERIES) against prepared standards of known concentration. Then, Cd removal efficiency of bacterial isolate was also measured using the following formula.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePollution load index, daily intake, and health risk assessment of Cd in animals in treatments\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTreatments\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePollution Load Index (PLI)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eDaily Intake of Metal (DIM)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e \u003cp\u003eHealth Risk Index (HRI)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCow\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSheep\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBuffalo\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCow\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSheep\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eBuffalo\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.037\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.221\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.192\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.252\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.156\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.134\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.176\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.002\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.006\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eR = (P0 \u0026ndash; Pe / P0) *100 (1)\u003c/p\u003e \u003cp\u003eIn this equation, R\u0026thinsp;=\u0026thinsp;the percentage of metal removal by the fungal biomass, P0\u0026thinsp;=\u0026thinsp;the\u003c/p\u003e \u003cp\u003einitial concentration of metal ions (ppm) and Pe\u0026thinsp;=\u0026thinsp;the final concentration of metal ions\u003c/p\u003e \u003cp\u003e(ppm) in the experimental media.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Experiment Setup and Climate Conditions\u003c/h2\u003e \u003cp\u003eThe botanical garden of Government College University in Lahore hosted a greenhouse study. According to the Pakistan Meteorological Department (PMD), the region received an annual rainfall of 628 mm, with a maximum average temperature of 40\u0026deg;C and a minimum average temperature of 27\u0026deg;C. \u003cem\u003eZea mays\u003c/em\u003e L. (Maize) seeds were sown in pots, and after 10 days of growth, urea and diammonium phosphate (DAP) were applied. Si was used as a Si source. Similarly, 8 treatments were included in the experiment: T1\u0026thinsp;=\u0026thinsp;Control; T2\u0026thinsp;=\u0026thinsp;2mM Silicon T3\u0026thinsp;=\u0026thinsp;Bacteria; T4\u0026thinsp;=\u0026thinsp;2mM Silicon\u0026thinsp;+\u0026thinsp;Bacteria; T5\u0026thinsp;=\u0026thinsp;Cd (10ppm); T6\u0026thinsp;=\u0026thinsp;2mM Silicon\u0026thinsp;+\u0026thinsp;Cd (10ppm); T7\u0026thinsp;=\u0026thinsp;Bacteria\u0026thinsp;+\u0026thinsp;Cd (10ppm); T8\u0026thinsp;=\u0026thinsp;2mM Silicon\u0026thinsp;+\u0026thinsp;Bacteria\u0026thinsp;+\u0026thinsp;Cd (10ppm). Values were the average of three replicates\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SE). These treatments were applied in pots with 3 kg of soil. The physicochemical characteristics of Bhal soil were [Soil texture (Clay loam), sand (38%), silt (32%), clay (36%), organic matter (0.86%) (Estefan et al., 2013), pH (7.48) (Walkley et al., 1947), EC (1.37 dSm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Hailegnaw et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and CEC (5.96 cmol Kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) (Robertson et al., 1999)]. Six to seven seeds were sown in each pot, and approximately five to six seeds germinated after 4 to 10 days. The plants were harvested after 40 days. Roots and shoots were carefully removed and washed with distilled water to remove soil particles. Both plant and soil samples were stored in labelled zipper bags and immediately stored for further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Fodder Quality Properties\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1 Measurement of Relative Water Content and Chlorophyll\u003c/h2\u003e \u003cp\u003eRelative water content (RWC) was measured by cutting a small piece of leaf. The fresh weight of the leaf was recorded, after which it was placed in a beaker filled with water for 4 hours. Following this, the leaf was reweighed to determine its turgid weight. The leaf was then placed in an oven for 24 hours to obtain its dry weight. RWC was calculated using the following formula:\u003c/p\u003e \u003cp\u003eRWC = (FW- DW) / (TW-DW)\u003c/p\u003e \u003cp\u003eFW\u0026thinsp;=\u0026thinsp;leaf fresh weight\u003c/p\u003e \u003cp\u003eDW\u0026thinsp;=\u0026thinsp;leaf dry weight\u003c/p\u003e \u003cp\u003eTW\u0026thinsp;=\u0026thinsp;leaf turgid weight\u003c/p\u003e \u003cp\u003eChlorophyll content was determined using the method described by Strain et al. (1996). Fresh leaves 0.1 grams were ground with 80% acetone in a pestle and mortar. After grinding, we used acetone to increase each sample's volume to 10 mL. The samples were then centrifuged for 5 minutes at 4000 rpm. The absorbance of the supernatant was measured using a UV/Visible spectrophotometer (Spectro scan 80D, Kyoto, Japan) at wavelengths of 663 nm and 645 nm to determine chlorophyll content.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2 Determination of Secondary Metabolites\u003c/h2\u003e \u003cp\u003eTotal phenolics were measured using the method described by Sultana et al. (2012). To determine total phenolics, 0.1 g of shoot tissue was ground with 4 mL of acetone. A small amount (60 \u0026micro;L) of the sample extract was mixed with 4,740 \u0026micro;L of distilled water, 300 \u0026micro;L of Folin-Ciocalteu reagent, and 900 \u0026micro;L of Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e. The prepared mixture was incubated at 55\u0026deg;C, then cooled, and its absorbance was measured using a UV/Visible spectrophotometer (Shimadzu UV 1201, Kyoto, Japan) at 760 nm. Protein content was determined using the Bradford (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1976\u003c/span\u003e) method. Briefly, 0.1 g of shoot tissue was ground in a pestle and mortar. A 200 \u0026micro;L aliquot of each sample was diluted with 1800 \u0026micro;L of distilled water, followed by the addition of 2 mL of Bradford reagent. The mixture was then incubated for 15\u0026ndash;20 minutes at 80\u0026deg;C. After incubation, absorbance was measured at 595 nm using a UV/Visible spectrophotometer (Spectro scan 80D, Kyoto, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e\u003cb\u003e2.4.3 Measurement of MDA, ROS Production and Antioxidant Enzyme Assay\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eMDA (malondialdehyde) content was estimated using the method of Kanwal et al. (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Shoots (0.5 g) were ground with 5 mL of 20% trichloroacetic acid (TCA) solution. The mixture was centrifuged (HERMLE Z167M) at 10,000 rpm for 10 minutes. 2.5 mL of the supernatant, 1 mL of 0.5% (w/v) TCA and thiobarbituric acid (TBA) were added. This mixture was heated for 30 minutes at 95\u0026deg;C and then cooled in an ice bath. The absorbance was measured at 532 nm and 600 nm using a spectrophotometer (Spectro scan 80D, Kyoto, Japan). MDA content was calculated as the difference in absorbance between 532 nm and 600 nm using Lambert's equation. Hydrogen peroxide (H₂O₂) was determined by grinding 0.5 g of fresh maize shoots with 5 mL of 0.1% TCA solution, followed by centrifugation at 12,000 rpm for 15 minutes. After centrifugation, 1 mL of supernatant was mixed with 1 mL of 10 mM potassium phosphate (K-P) buffer (pH 7.0) and 2 mL of 1M potassium iodide (KI) solution. The absorbance of the mixture was measured at 390 nm to calculate the H₂O₂ concentration (Latef et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Fresh maize shoots (0.6 g) were ground up in a buffer solution (0.2 M potassium phosphate, pH 7.8) that also had 0.1 mM EDTA in it. This was used to make plant extracts. The mixture was centrifuged (HERMLE Z167M) at 15,000 rpm for 20 guaiacol solution and 30 \u0026micro;L of 12 mM H₂O₂. The absorbance was checked at 436 nm using a spectrophotometer (Shimadzu UV-1201, Kyoto, Japan) (Sharma et al., 2017).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.4.3. Maize fodder yield and Cd tolerance ability\u003c/h2\u003e \u003cp\u003eFresh maize shoots were washed with distilled water to remove dust and soil particles. The shoots and roots were air-dried for 48 hours and then oven-dried at 70\u0026deg;C to achieve a constant weight. The weight of each sample was recorded. To find out how much cadmium (Cd) was in the shoots and roots, 0.5 g of maize samples were broken down in a dilute acid solution of 3 parts HNO\u003csub\u003e3\u003c/sub\u003e and 1 part HClO\u003csub\u003e3\u003c/sub\u003e at 180\u0026deg;C until clear fumes came out. Following the digestion process, we added distilled water to reach a total volume of 50 mL and then double-filtered the mixture to obtain a clear solution. Standards for Cd were prepared according to Jones et al. (1990), and the Cd concentration was measured using a multi-sequential Atomic Absorption Spectrophotometer (iCE 3000 SERIES). For soil samples, 1 g of soil was digested using the same di-acid mixture (HNO₃: HClO₄, 3:1) at 180\u0026deg;C until clear fumes were formed. After digestion, distilled water was added to make the final volume 50 mL, followed by double filtration to obtain a clear solution. Standards of Cd were prepared, and the concentration of Cd in soil samples was determined using the same multi-sequential Atomic Absorption Spectrophotometer (iCE 3000 SERIES) (Jones et al., 1990).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Health risk assessment for grazing animals\u003c/h2\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1 Pollution load index\u003c/h2\u003e \u003cp\u003eIt is used to determine the contamination of heavy metals in soil. Its formula given by Liu et al. (2005) is:\u003c/p\u003e \u003cp\u003ePLI\u0026thinsp;=\u0026thinsp;Metal concentration analysed in soil samples /Metal reference value in soil\u003c/p\u003e \u003cp\u003eThe reference value for Cd is 70 mg/kg (Singh et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.5.2 Daily intake of metals\u003c/h2\u003e \u003cp\u003eThe formula to find the daily intake of metals (DIM) is:\u003c/p\u003e \u003cp\u003eDIM\u0026thinsp;=\u0026thinsp;Analysed metal concentration in forage*Conversion Factor*Daily food intake/Average body weight of animals\u003c/p\u003e \u003cp\u003eFor the conversion factor, value was 0.085 (Jan et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). For cows, daily intake of food was 12kg, and average body weight is 200 kg, whereas the daily intake and average weight of sheep were 1.3kg and 75kg, respectively (Johnsen and Aaneby \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). For buffaloes, average weight was 300 kg, and their daily intake was 12.5kg (Briggs and Briggs, 1980).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.5.3 Health risk index\u003c/h2\u003e \u003cp\u003eOral reference dose for Cd is 0.017 mg/kg/day (USEPA 2020), and the formula to find the HRI is:\u003c/p\u003e \u003cp\u003eHRI\u0026thinsp;=\u0026thinsp;Daily intake of metal/Oral reference dose\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Statistical Analysis\u003c/h2\u003e \u003cp\u003eThis experiment was performed in a completely randomized design and one way ANOVA by using Microsoft Excel 2013\u0026reg; (Microsoft cooperation, USA) and Statistix 8.1\u0026reg; (Analytical software, Tallahassee, USA), XL STAT for principal component analysis (PCA), was applied to analyze the results. MEGA 11 was used to prepare phylogenetic trees. The least significant design (LSD) was applied to compare the means of two separate groups. The results showed the mean of three replicates (n\u0026thinsp;=\u0026thinsp;3) with standard error \u0026plusmn; (SE).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Isolation and Identification of Rhizobacteria\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eIn this study, bacteria were isolated from rhizosphere of wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e) and subjected to morphological analysis and characterization (Supplementary Table\u0026nbsp;1). The bacterial strain known as SN215, which showed a probable potential for metal resistance, was gram positive. Taxonomic classification of the mentioned strain was done using sequence homology analysis and phylogenetic evaluation. A Basic Local Alignment Search Tool (BLAST) analysis was performed on 16S rRNA sequence and classified SN215 as SN215.\u003c/p\u003e \u003cp\u003eTo further confirm this homology, the relevant sequences from similar strains were retrieved from The National Center for Biotechnology Information (NCBI) database, and a phylogenetic tree was constructed using MEGA X, which demonstrated that SN215 clustered in close proximity to SN215 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The strain was sequenced on the bases of 16S rRNA gene, and sequence was successfully submitted to GenBank under the accession ID SUB13767386 with the nucleotide accession number OR453182.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Biosorption Trial of SN215\u003c/h2\u003e \u003cp\u003eOur results indicated that SN215 exhibited the highest biosorption efficiency 83%, at medium cadmium concentration. Based on these findings, the medium Cd concentration was selected for further investigation in pot experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), where the performance of bacteria in enhancing Cd tolerance of maize fodder was evaluated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Maize Fodder Growth and Yield\u003c/h2\u003e \u003cp\u003eThe combined application of bacterial strain SN215 and Si prominently improved the shoot biomass under Cd stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Our data depicted that plants of T8 demonstrated a remarkable increase in the shoot fresh biomass (two-folds) and shoot dry biomass (two-folds) compared T5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). However, the applied Cd stress significantly influenced the growth parameters. T5 significantly reduced the shoot fresh weight (SFW) of maize (45.38%) compared to T1. A similar trend was observed for shoot dry weight of maize plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Moreover, T5 significantly (one-fold) reduced the shoot dry weight (SDW) of maize compared to T1. It is obvious from the figure that, irrespective of Cd toxicity in soil, different applied treatments enhanced the morphological traits of plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Maximum increase (two-folds) in SFW was reported with treatment T8 relative to T5. Similarly, the SDW of maize plant for Cd toxicity also showed a similar trend with maximum SDW up to (two-fold) in treatment T8 as compared to T5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Maize Fodder Quality\u003c/h2\u003e \u003cp\u003eRWC exhibited significant variation across all treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The RWC of maize was significantly reduced (29%) under 10 ppm Cd as compared to control plants. Remarkably, the combination of Si and SN215 under cadmium stress (T8) helped to restore RWC to around 60%, mitigating the negative impact of cadmium stress by approximately 16% compared to T5. Total phenolic content varied significantly under different treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The control plants (T1) exhibited phenolic levels of about 85 \u0026micro;g g⁻\u0026sup1;, while plants receiving the combined treatment of Si\u0026thinsp;+\u0026thinsp;SN215 had the highest phenolic content of 53% increase over the control. Under 10 ppm Cd stress, the phenolic content decreased to 23% compared to T4. However, plants treated with both Si and SN215 under Cd stress maintained phenolic content at 10% recovery compared to T5. The greenness of maize fodder varied significantly under different treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The highest chlorophyll content was recorded in T8, showing six-folds, nine-folds increase in chlorophyll a and chlorophyll b, respectively, as compared to T5. 10 ppm Cd stress led to a significant decline in chlorophyll content of 89%, 92.8% reduction in chlorophyll a and chlorophyll b respectively compared to T1. Total protein content exhibited a substantial variation among treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). T1 showed a protein concentration of approximately 18 mg g⁻\u0026sup1;. 10 ppm Cd stress led to a significant decline in protein content of 60% reduction compared to T1. Impressively, plants of T8 showed 67% increase in protein concentration compared to T5.\u003c/p\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003e3.4.1 Cd Tolerance Capacity of Maize Fodder\u003c/h2\u003e \u003cp\u003eApplication of Cd stress significantly increased the maize plant's oxidative stress damage, as measured by the level of MDA (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). T5 reported a maximum increase in shoot MDA (up to one-fold) compared to T1. However, the co-application of Si and SN215 treatment decreased the shoot oxidative stress under Cd stress as measured by oxidative stress marker MDA. A maximum decrease (61.96%) in shoot MDA was reported with treatment T8 relative to T5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Moreover, application of Cd stress also increased H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration in maize plants, adding to oxidative stress burden of treated plants. Compared to control plants, T5 showed a maximum five-fold increase in shoot H2O2 concentration. However, conjoined application of Si and SN215 decreased the shoot oxidative stress against the increasing levels of Cd toxicity in soil. T8 reported the maximum decrease (59.43%) in shoot H2O2 concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Our results clearly demonstrate that combined application of Si and SN215 predominantly alleviated oxidative stress of maize plants under Cd stress, conferring tolerance stress-treated plants against Cd. Data on the activities of antioxidant enzymes revealed that presence of Cd stresses significantly inhibited the functioning of APX and CAT in treated plants compared to control ones (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Under 10 ppm Cd toxicity, the concentrations of shoot APX (88.2%) and CAT (67%) were significantly lower than the control. When compared with T1, a three-fold increase in shoot APX as well as CAT was obvious when maize plants were subjected to Si\u0026thinsp;+\u0026thinsp;SN215, under 10 ppm Cd stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), demonstrating stimulatory effect of Si and SN215 combination on activities of these antioxidant enzymes. The concentrations of shoot SOD (67.11%) and shoot POD (79.18%), among other pillars of antioxidant enzymes, were significantly lower under Cd stress compared to control. However, the application of Si and SN215 enhanced the concentrations of mentioned enzymes in maize plants under Cd toxicity. Maximum increase was observed in shoot SOD (71%) and shoot POD (three- fold) with T8 as compared to T5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003e3.4.2. Cd Contents in Soil and Shoots of Maize Fodder\u003c/h2\u003e \u003cp\u003eIn control plants, Cd uptake was negligible, while it peaked at 2.6 mg g⁻\u0026sup1; DW in T5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), marking a 13-fold increase compared to the T1. In T6, Cd uptake was reduced by 30%, while in T7, it was lowered by 42%. Notably, in T8 Cd uptake decreased by 95% compared to T5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Regarding soil Cd concentration, the concentration remained low in the non-Cd-stressed treatments (T1-T4), but it was maximum in the presence of Cd stress (T5) (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eT8 led to a decrease in Cd toxicity to 30% as compared to T5, demonstrating its effectiveness in decreasing the bioavailability of Cd in the soil. The proposed study examined translocation factors (TF) and bioconcentration (BCF) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). This figure shows that overall Cd (TF) concentrations were less than 1, effectively preventing TF from root to shoot. These results showed that the roots of maize plants acquire higher total Cd ions than the shoots. The influence of maize plant capillary action on the transfer of total Cd is another basis for the TF factor (Takarina and Pin, 2017).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003e3.4.3. Health Risk Assessment for Grazing Animals\u003c/h2\u003e \u003cp\u003eAs a general trend, in treatments T1-T4, the value of PLI i.e., the level of metal contamination was consistently zero, indicating no detectable pollution load. HRI, DIM, and PLI from T1 to T4 can be declared as 'no-risk', 'safe' or 'low-risk', hence posing no adverse health effects to the grazing animals (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). However, the highest PLI was observed in treatment T5 (0.037), followed by T7 (0.015), T6 (0.013), and T8 (0.009), suggesting that these treatments were associated with varying levels of metal Contamination. The highest Cd contamination in T5 calls for concern, particularly among people who consume animal products. In terms of metal intake, cows, sheep, and buffalo showed no daily intake of metal for treatments T1-T4, indicating the absence of metal exposure in these treatments (Table\u0026nbsp;2). Treatment T5 exhibited the highest DIM values for all three species (0.003 for cows, sheep, and buffalo), indicating significant metal ingestion. The metal intake in treatments T7 and T8 was negligible, as indicated by very low DIM values (0.000). Likewise, highest HRI was observed with T5 (0.221 for cows, 0.192 for sheep, and 0.252 for buffalo), suggesting the greatest health risk, especially for buffalo. The low HRI values observed in treatments T7 and T8 (T7: 0.004 for cows, 0.003 for sheep, 0.002 for buffalo; T8: 0.006 for cows, 0.005 for sheep, 0.006 for buffalo) indicated minimal health risks.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Principle Component Analysis (PCA)\u003c/h2\u003e \u003cp\u003ePhysiological and biochemical parameters of maize were analyzed by constructing biplots of PCA to evaluate the most efficient treatment in study. PCAs contributed to 94.34% variance in plant biplots (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Following that, proteins had a strong positive relationship with CAT, APX, SOD, and POD but a strong negative relationship with H2O2 and MDA (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Similarly, Cd showed a highly negative correlation with proteins, CAT, phenolics, APX, SOD, and POD, while Cd showed positive correlation with H2O2 and MDA. The PCA results showed that addition of Si and SN215 improved antioxidant enzymes and secondary metabolites in maize plants. The PCA results significantly investigated that the application of T8 has the greatest effect on growth and biochemical parameters of maize crops under cadmium stress.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Biosorption Trial on SN215\u003c/h2\u003e \u003cp\u003eIn our study, biosorption trial with cadmium concentrations of 5 ppm (low), 10 ppm (medium), and 15 ppm (high), biosorption efficiency was calculated after 24 hours and 48 hours, it was shown that biosorption effectiveness peaked at 10 ppm. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The result fits with what we know about how the concentration of heavy metals in a solution can affect biosorption processes. This is because higher concentrations may make binding sites on the surface of bacteria cells full, which makes biosorption less effective at high levels. A study found that extending the contact period beyond 24 hours reduced the effect size and removal efficiency for Cd (II). The increased toxicity of the metal ions to living bacterial strains may cause a decrease in biosorption capacity with longer contact time (Ibuot et al., 2017; Igiri et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Contact lengths longer than 24 hours can impact the metal speciation, solubility, and eventual desorption or precipitation of metal ions, while the metabolic activities of living bacteria can cause pH variations in the metal solution (Ratzke et al., 2018; Xie et al., 2018). At 10 ppm, the biosorption efficiency remained steady, indicating that this dosage is optimum for the bacterial strains studied. The abundance of binding sites and the maintenance of the bacteria's metabolic activity without the inhibitory effects observed at higher concentrations may be responsible for this stability (Hossain and Aditya, 2015). The results show that the bacterial strains used in the biosorption studies are very good at absorbing cadmium at low to moderate levels. This is important for making bioremediation methods that work well (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Effect of SN215 on Maize Fodder Growth and Yield\u003c/h2\u003e \u003cp\u003eThe growth of maize fodder was significantly reduced because of phytotoxic nature of Cd, especially affecting the shoot fresh and dry weights (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Biomass serves as a basic ecological parameter to determine plant growth and reflects the plants' ability to manage nutritional reservoirs and provide necessary data to evaluate the plant's potential under stressful conditions (Cid et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Cadmium aggregation reduces seed germination, primary seedling growth, and overall plant biomass. It alters the rate of photosynthesis, transpiration rate, relative water content, electrolyte leakage and stomatal conductance. Cd stress switches on the production of ROS, which leads to chromosomal abnormalities, genetic mutations, and DNA impairment, hence affecting the cell division as well as cell cycle (El Rasafi et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eShah et al. have demonstrated that Cd stress significantly reduced fresh as well as dry biomass in maize (Shah et al., \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Our results demonstrate that application of Si alone or in combination with Rhizobacteria is a substantial mitigation of Cd stress, with a two-fold increase in shoot fresh weight when Si was applied along with Rhizobacteria. Si likely improved plant tolerance by strengthening cell walls, while Rhizobacteria may have enhanced nutrient uptake or immobilized Cd in the soil. These results suggest that combining Si and Rhizobacteria can be an effective strategy for improving crop growth under heavy metal stress.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Effect of Si and SN215 on Maize Fodder Quality\u003c/h2\u003e \u003cp\u003eOne of the many reasons for Cd-triggered growth inhibition may be associated with decreased relative water content in plants stressed with Cd, as reported previously (Kaya et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In this experiment, the combined application of Si and Rhizobacteria significantly improved the RWC of Cd-stressed plants, indicating that the treatment helped maize fodder plants maintain turgor pressure even in the presence of Cd toxicity. Our results are analogous to those of Azizi et al, where improvement in relative water content of savory plants improved plant biomass under Cd stress (Azizi et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Heavy metals hinder photosynthesis, affecting the generation of chlorophyll and carotenes. Moreover, HMs stress affects the activities of key enzymes involved in photosynthesis. Most importantly, HM stress causes the down-regulation of light-harvesting compounds chlorophyll and chlorophyll b, restricting photosynthesis and ultimately affecting plant development (Zhou et al., \u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Likewise, in the present study, the concentration of chlorophyll content (chlorophyll a and b) was reduced significantly with applied stress under T4 (10ppm Cd) as compared to control T1. However, the combined application of Si and SN215 significantly improved photosynthetic pigments (chlorophyll a, chlorophyll b and xanthophyll) of maize plants. The synergistic effect of Si and SN215 probably enhanced the photosynthetic pigments in maize by improving nutrient uptake, strengthening the plant structure, protecting plants against oxidative stress, and stimulating growth-promoting processes. This synergistic effect resulted in better overall plant health and higher photosynthetic efficiency. The improved nutrient uptake, water status, and hormonal balance in the plant could be the cause of the increased photosynthesis rate. Our results are in parallel with studies carried out by Cipriano et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Koll\u0026aacute;rov\u0026aacute; et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2019\u003c/span\u003e and Sarathambal et al., \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2017\u003c/span\u003e in which structure of chloroplast was improved using Si and SN215 under Cd stress. Proteins and phenolic compounds play an important role in maintaining proper structure and function of plant cells. All the important biochemical reactions are liked with proper functioning of protein and phenolics (Goncharuk et al., 2023). According to previous reports, Cd has the potential to reduce the overall protein content of plants by restricting the uptake of potassium and magnesium, promoting post-translational modifications, reducing the rate of photosynthesis, enhancing protein degradation, and prohibiting RuBisCo activity (Monteiro et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Under Cd stress, the concentration of phenolic compounds, known as metal-chelating agents, increases (Goncharuk and Zagoskina, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, the presence of Cd stress significantly reduced the concentration of proteins and phenolics (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), potentially due to Cd-generated toxicity. The application of Si and Lysinibaciluus sp. significantly improved the concentration of proteins and phenolics in T7 compared to Cd stress (T4) alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Similar results have been reported in previous studies where individual applications of Si and SN215 have helped HM-stressed plants combat heavy metal stress by increasing total protein and phenolic content (Mihaličov\u0026aacute; Malčovsk\u0026aacute; et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Anwaar et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Zheng et al., \u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e4.4 Effect of Si and SN215 on Cd Tolerance Capacity of Maize Fodder\u003c/h2\u003e \u003cp\u003eHeavy metal stress disturbs the dynamic equilibrium of ROS production in plants. These ROS start accumulating in membranes of plants, which disturbs structure and functions of cell membranes. It has been documented that high level of Cd can cause production of malondialdehyde (MDA) and hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), which are indicators of cell membrane damage (Deng et al., 2024). Our research showed that Cd stress made oxidative damage much worse in maize plants. This was shown by high levels of MDA and H2O2, which are signs of lipid peroxidation and oxidative stress. A one-fold increase in MDA and a five-fold increase in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was observed under 10 ppm Cd stress. However, the application of Siand SN215 effectively mitigated oxidative damage, reducing MDA by 61.96% and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e by 59.43%. There are two ways by which Cd-generated oxidative stress burden can be mitigated: enzymatic and non-enzymatic antioxidant defence systems, which can maintain redox homeostasis. The enzymatic defence antioxidants enzymes such as ascorbate peroxidase (APX), catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), and dehydroascorbate reductase (DHAR). The non-enzymatic pathway scavenges ROS with the help of redox or antioxidant metabolites, such as glutathione (GSH), ascorbate (AsA), nicotinamide adenine dinucleotide phosphate (NADPH) to lighten the oxidative stress (Jung et al., 2020). In the current study, cadmium stress (10 ppm) significantly inhibited the activities of key antioxidant enzymes, including APX, CAT, SOD, and POD in maize. This reduction in enzyme activity reflects the disruption of the plant's ability to mitigate ROS generated under Cd stress. The big drop in shoot APX (88.2%) and CAT (67%) suggests that oxidative stress caused by Cd makes it much harder for cells to get rid of hydrogen peroxide and other ROS, which can damage cells. The interactive role of Si and PGPBs may alleviate the oxidative damage in numerous plant species via upregulation of ROS-scavenging enzymes along with non-enzymatic branch of antioxidation (Kim et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Verma et al., \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Interestingly, in our current study, Cd-stressed plants, when inoculated with SN215 as well as supplied with Si, the activities of antioxidant enzymes were significantly increased compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), highlighting the potential of Si and SN215 to bolster the antioxidant defense system in maize under Cd stress. Our results are clearly in line with those of Farooq et al. where application of Si effectively mitigated the deleterious effects of Cd stress in cotton by decreased production of MDA and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e hence reducing net oxidative stress burden on the plants (Farooq et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Our results are also in parallel with studies of Luyckx et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Das et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e in which growth of \u003cem\u003eCannabis sativa\u003c/em\u003e L. and rice ( Japonica and Indica) was improved by decreasing the concentration of ROS using Si and SN215 under heavy metal stress.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003e4.5 Effect of Si and SN215 on Cd Contents in Soil and Shoots of Maize Fodder\u003c/h2\u003e \u003cp\u003eCd is extremely mobile once inside the plant tissues and greatly impacts physiological growth (Saidi et al., \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In the current study, the concentration of Cd significantly increased with applied stress (10ppm Cd) compared to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). However, the application of Si and SN215 proved to be beneficial in reduction of bioavailability of heavy metals to plants by different possible mechanisms. Si causes immobilization of heavy metals (Sahebi et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Soil pH plays an important role in immobilization of heavy metals by formation of silicate complexes. Heavy metals contaminated soils, which are amended by Si\u0026thinsp;+\u0026thinsp;Cd is absorbed by oxides of iron and manganese. So, bioavailability of heavy metals can be limited to plants due to formation of silicate complexes (Ma et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Photosynthesis is a crucial physiological process for plants, and as per numerous reports, this process is severely inhibited by Cd stress. The rate of photosynthesis inhibition is directly proportional to the concentration of Cd (K\u0026uuml;pper et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Consistent with previous studies, the application of Cd caused a drastic reduction in the chlorophyll a and b content (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The reduction in chlorophyll content was possible because of inhibition of synthesis of chlorophyl due to Cd toxicity. However, the combined application of Si and SN215 significantly improved photosynthetic pigments of maize plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The observations made in the current study are in accordance with previous reports where application of Si minimized the damage to chlorophyll content in \u003cem\u003eZea mays\u003c/em\u003e plants under Cd toxicity (Saleem et al., \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and application of plant growth promoting bacteria (\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e and \u003cem\u003eBurkholderia gladioli\u003c/em\u003e ) in \u003cem\u003eLycopersicon. esculentum\u003c/em\u003e seedlings improved chlorophyll content of seedlings under Cd stress as compared to control ones (Khanna et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The increased under Cd stress chlorophyll content in our study after application of Si and SN215 is perhaps due to Cd chelation by Si and SN215, hence restricted uptake of Cd in plants. Moreover, inoculation of plants with bacteria might have enhanced the nutrient uptake, improving photosynthetic potential of plants under Cd toxicity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003e4.6 Effect of Si and SN215 on Health Risk Assessment for Grazing Animals\u003c/h2\u003e \u003cp\u003eEstimation of exposure level of heavy metals, along with tracking their route of contamination for target organisms, is crucial for comprehension of associated health risks (Li et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Health risks linked with atmospheric contamination possibly arise from the inhalation of heavy metal particles or from consuming polluted food (Xiong et al., \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Shahid et al., \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The pollution load index (PLI) results for Cd metal ranged from 0.427\u0026ndash;0.805 in the soil samples (Table\u0026nbsp;2). T8 practised the minimum value, while T5 represented the maximum value. Overall, the values of pollution load index were observed to be less than 1, which indicated that all the treatments are unpolluted (Khan et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The values reported by Ezemokwe et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and Khan et al. (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) were 0.05 and 0.0649 mg/kg, which are observed to be lower than the observed concentration in our studies. The observed PLI range was less than 1 in all treatments, which represents that all treatments reduced Cd toxicity in present research. DIM (daily intake of metal) presents an index for determination of body exposure to heavy metal through feeding (Daniele et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The daily Cd intake varied from 0.00 to 0.004 mg/kg/day. The study focused on the minimum intake in sheep, while the buffaloes grazing on Zea mays perceive the maximum concentration. The HRI value for Cd amounted to 0.00 mg/kg/day and was 0.252 mg/kg/ day. The maximum value of HRI was analysed in the buffaloes, while the minimum level was assessed in the sheep (Table\u0026nbsp;2). Similar results were observed by Khan et al. (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), who conducted a pot trial to assess the impact of wastewater on zinc accumulation in various forages and their associated health risks. They grew both summer forages (e.g., Zea mays, Sorghum bicolor) and winter forages (e.g., Trifolium alexandrinum, Brassica napus) using sewage and tap water treatments. Results showed that the pollution load index, daily intake of metal, and health risk index for zinc were all below 1, indicating that the consumption of these forages posed no health risk. This study showed the DIM range (0.00164\u0026ndash; 0.0813mg/kg/day), which is like present results.\u003c/p\u003e \u003c/div\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eSilica with SN215 showed efficient performance in reducing cadmium toxicity in \u003cem\u003eZea mays\u003c/em\u003e crop through scavenging burst production of ROS, stimulating the enzymatic and non-enzymatic defence mechanisms, and improving photosynthetic machinery of experimental plants. Hence application of silica in combination with SN215 has proved to be an eco-friendly soil conditioner strategy that can be used in developing countries to limit the deleterious effects of Cd on fodder crops. Overall, the data indicated that treatment T5 poses the most significant health concerns due to its higher pollution load, metal intake, and associated health risks, particularly for buffalo. Treatment T6 also posed a moderate risk, while treatments T7 and T8 showed only minimal impact.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSeemal Naeem:\u0026nbsp;\u003c/strong\u003eWriting – review \u0026amp; editing, Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Conceptualization. \u003cstrong\u003eWaqas ud Din Khan:\u0026nbsp;\u003c/strong\u003eFormal analysis, Conceptualization, Investigation, Resources, Project administration. \u003cstrong\u003eTahmina Nazish:\u0026nbsp;\u003c/strong\u003eWriting – review \u0026amp; editing, Supervision, Methodology, Funding acquisition, Formal analysis, Conceptualization. \u003cstrong\u003eMuhammad Umer Farooq Awan:\u0026nbsp;\u003c/strong\u003eFormal analysis, Writing – review \u0026amp; editing, Resources. \u003cstrong\u003eUsman Ijaz:\u0026nbsp;\u003c/strong\u003eWriting – review \u0026amp; edit- ing, Supervision, Methodology, Funding acquisition, Formal analysis, Conceptualization.\u003cstrong\u003e\u0026nbsp;Abdul Sattar Nizami:\u0026nbsp;\u003c/strong\u003eWriting – review \u0026amp; edit- ing, Supervision, Funding acquisition, Formal analysis. \u003cstrong\u003eMohsin Tanveer:\u0026nbsp;\u003c/strong\u003eWriting – review \u0026amp; editing, Supervision, Funding acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAhmad P, Abdel Latef AA, Abd_Allah EF, Hashem A, Sarwat M, Anjum NA, Gucel S (2016) Calcium and potassium supplementation enhanced growth, osmolyte secondary metabolite production, and enzymatic antioxidant machinery in cadmium-exposed chickpea (Cicer arietinum L). 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Plant Signal Behav 16(1):1836884. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.1016/j.jhazmat.2020.123608\u003c/span\u003e\u003cspan address=\"10.1016/j.jhazmat.2020.123608\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cleaner production, Sustainability, Wastewater, Animal fodder, Cd stress","lastPublishedDoi":"10.21203/rs.3.rs-6592002/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6592002/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCadmium (Cd) enrichment in agricultural soils due to wastewater irrigation poses significant risks to the growth, quality, and yield of maize fodder. This study explores the role of SN215 and silicon (Si) in mitigating cadmium (Cd) toxicity in maize fodder and reducing associated health risks in grazing animals. The SN215 strain, isolated from the wheat rhizosphere and identified as SN215, exhibited 83% Cd biosorption efficiency at a medium Cd concentration (10 ppm). Under controlled greenhouse conditions, the combined application of SN215 and Si significantly enhanced maize growth, resulting in a two-fold improvement in shoot fresh and dry biomass. Furthermore, the treatment improved relative water content (RWC), phenolic levels, chlorophyll concentrations, and protein content, restoring RWC to 60% and increasing phenolic content by 10% in comparison to plants under Cd-only stress. The treatment significantly increased antioxidant enzyme activities (ascorbate peroxidase, catalase, superoxide dismutase, and peroxidase) while reducing oxidative stress markers like malondialdehyde and hydrogen peroxide by 61.96% and 59.43%, respectively. Moreover, the combined application of SN215 and Si reduced Cd uptake in shoots by 95% and soil Cd levels by 30%. Health risk assessments revealed a negligible daily intake of metals and a health risk index for grazing animals with SN215 and Si treatment, highlighting its effectiveness in mitigating Cd toxicity. The findings demonstrate the potential of SN215 and Si co-application as an eco-friendly strategy to improve fodder quality and reduce health risks in Cd-contaminated environments.\u003c/p\u003e","manuscriptTitle":"Unravelling Potential of Silicon and Rhizobacteria in Reducing Cd-related Health Risk in Grazing Animals by Enhancing Maize Fodder Quality","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-13 09:53:57","doi":"10.21203/rs.3.rs-6592002/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8b73e39f-07c1-43df-b086-893501328183","owner":[],"postedDate":"May 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-05-14T19:19:54+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-13 09:53:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6592002","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6592002","identity":"rs-6592002","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}