Modulation of Cd carriers by innovative nanocomposite (Ca+Mg) and Cd-resistance microbes (Bacillus pumilus): a mechanistic approach to enhance growth and yield of rice (Oryza sativa L.)

Cadmium (Cd) is a well-known pollutant in agricultural soil, affecting human health through the food chain. To combat this issue, Ca + Mg (25 mg L−1) nanocomposite and Bacillus pumilus, either alone or combined, were applied to rice plants under Cd (5 mg kg−1, 10 mg kg−1) contamination. In our study, growth and yield traits demonstrated the beneficial influence of Ca + Mg and B. pumilus application in improving rice defense mechanism by reducing Cd stress. Combined Ca + Mg and B. pumilus application increased SPAD (15), total chlorophyll (18), chlorophyll a (11), chlorophyll b (22), and carotenoids (21%) with Cd (10 mg kg−1), compared to the application alone. Combined Ca + Mg and B. pumilus application significantly regulated MDA (15), H2O2 (13), EL (10), and O2 •– (24%) in shoots under Cd (10 mg kg−1), compared to the application alone. Cd (10 mg kg−1) increased the POD (22), SOD (21), APX (12), and CAT (13%) in shoots with combined Ca + Mg and B. pumilus application, compared to the application alone. Combined Ca + Mg and B. pumilus application significantly reduced Cd accumulation in roots (22), shoots (13), and grains (20%) under Cd (10 mg kg−1), compared to the application alone. Consequently, the combined application of Ca + Mg and B. pumilus is a sustainable solution to enhance crop production under Cd stress.


Introduction
Heavy metal contamination, such as cadmium (Cd), is a widespread environmental hazard that has gained significant attention in agricultural soil (Alengebawy et al., 2021;Ali et al., 2022a;Zulfiqar et al., 2023a).Cd is rated 7th on the list of hazardous compounds due to its carcinogenic properties, according to the US Agency for Toxic Substances and Disease Registry (ASTDR, 2021;Zulfiqar et al., 2022a).Due to inadequate crop production strategies, Cd has caused food safety issues in recent years.Plants rapidly uptake, accumulate, and translocate Cd into different parts, particularly edible ones, triggering food safety issues (Liu et al., 2010;Zeb et al., 2022;Zulfiqar et al., 2022b).Rice is a staple crop with an annual production of 754.6 million tons, feeding half of the world's population and representing a vital component of the global agricultural economy (FAO, 2017).Cd toxicity inhibits plant height, root length, leaf area, and the number of leaves per plant in rice (Song et al., 2015).Cd significantly reduces the growth and agronomical attributes of rice (Wang et al., 2014;Li et al., 2022).Numerous studies reported that Cd stress enhances oxidative stress through reactive oxygen species (ROS) production in rice seedlings (Srivastava et al., 2014;Ayyaz et al., 2022;Nazir et al., 2022).Therefore, it is essential to develop an effective remediation strategy to deal with Cd-contaminated soil.Various physical, chemical, and biological remediation techniques have been adopted for the removal of heavy metal contamination.These techniques are often unrealistic due to poor efficiency (Nafees et al., 2018;Hou et al., 2020;Yang et al., 2020).Hence, there is a need to develop costeffective, eco-friendly, and innovative approaches to limit the impact of heavy metal pollution on global food safety (Chen and Li, 2018;Li et al., 2022).
Nanotechnology has garnered significant attention in the modern era, as this field has radically transformed modern science and is expanding exponentially (Bhardwaj et al., 2022).Recently, nanoparticles (NPs) have significantly enhanced plant growth and development by reducing heavy metal uptake in plants (Nafees et al., 2020;Babu et al., 2022;Ulhassan et al., 2022;Khalid et al., 2023;Nafees et al., 2024a).NPs mitigate stress by regulating phytohormones (Maqsood et al., 2023).According to a study, copper (Cu) NPs enhanced the growth and agronomical attributes of wheat by mitigating Cd toxicity (Noman et al., 2020).The foliar application of nano zinc (Zn) and iron (Fe) has shown a benefi cial effe cts in Rosmarinus offi cinalis (Hassanpouraghdam et al., 2020).Similarly, the foliar application of ceric oxide (CeO 2 ) and copper oxide (CuO) NPs enhanced the fresh weight, yield, and nutritional quality of cucumber (Hong et al., 2016).
Calcium (Ca) and magnesium (Mg) are ubiquitous metal elements in the Earth's crust, and their ionic forms (Ca 2+ and Mg 2+ ) in soil are vital plant nutrients.Mg plays a fundamental role in regulating physical and biochemical processes, and its deficiency is a limiting factor for crop production (Ishfaq et al., 2021(Ishfaq et al., , 2022)).Ca 2+ and Mg 2+ are absorbed by roots and transported to shoots via xylem.CaO NPs significantly enhanced plant biomass, and enzymatic and non-enzymatic antioxidative activity due to a substantial decrease in ROS species under Cd toxicity (Nazir et al., 2022).Li et al. (2022) reported that Ca 2+ and Mg 2+ treatment boosted rice growth and yield by reducing uptake and accumulation of Cd in grains, roots, stems, and leaves.Limited research has focused on investigating the influence of Ca and Mg NPs on plant-soil systems, particularly regarding soil microbial interactions with plants.
Like NPs, microbes play a vital role in plant growth by reducing the uptake and accumulation of heavy metals (Hassan et al., 2017;Ali et al., 2022b;Zulfiqar et al., 2023b).Bacillus pumilus is a growthpromoting bacterium in plants.B. pumilus reduces the uptake of cadmium and promotes plant height and photosynthetic pigments in rapeseed (Brassica napus L.) (Masood et al., 2020).Similarly, Bacillus spp.enhances the activity of ROS-scavenging enzymes such as POD, SOD, CAT, and APX, and boosts maize tolerance to Zn and Cu stress (Shahzad et al., 2021).Currently, no studies have been conducted on the combined effect of Ca + Mg nanocomposite and microorganisms on rice growth.Furthermore, the risk assessment of their toxicity to rice and soil is still in its early stages.Meanwhile, the combination of nanoparticles and microbial strain inoculation has recently attracted significant attention in agriculture due to their greater efficacy in alleviating heavy metal stress (Babu et al., 2022;Ahmed et al., 2023).
Therefore, a novel Ca + Mg nanocomposite was synthesized and the specific objectives of the current study were: a) to evaluate the individual and combined effect of Ca + Mg nanocomposite and B. pumilus on rice yield and growth; b) investigate the alleviating combined role of the Ca + Mg nanocomposite and microbes on oxidant and antioxidant enzymatic activity; c) to analyze the individual and combined effect of the Ca + Mg nanocomposite and B. pumilus on Cd uptake and accumulation in rice.Thus, the current study could offer an economically feasible alternative fertilizer that promotes sustainable agricultural crop production with higher nutritional value.

Soil collection and scrutiny
The soil was collected from the fields of the University of Agriculture, Faisalabad.Soil was air-dried and sieved through a 2 mm sieve.The soil used in this study was sandy clay loam according to Bouyoucos (1962).The electrical conductivity (EC) was 1.85 dS m −1 , and the pH (7.68) of the soil extract was measured using appropriate methods.Available Cd (0.07 mg kg −1 ) was measured following the standard procedure of Amacher (1996).Soil organic carbon was assessed by following the Walkley-Black protocol, while calcium carbonate, total nitrogen, available phosphorous, extractable potassium, and cation exchange capacity were 3.16 g kg −1 , 3.2%, 0.089%, 6.1 mg kg −1 , 92 mg kg −1 , 10.4 cmol (+) kg −1 , respectively, using appropriate methods.Similarly, Zn, Mn, and Fe were 5.13 mg kg −1 , 4.97 mg kg −1 , and 53.74 mg kg −1 , respectively, determined using the calcimeter method (Moodie et al., 1959;Jackson, 1962).

Ca + Mg nanocomposite synthesis
The calcium and magnesium nanocomposite was synthesized following the method of Das et al. (2018) using calcium nitrate tetrahydrate (Ca(NO 3 ) 2 • 4H 2 O) and magnesium nitrate tetrahydrate (Mg(NO 3 ) 2 • 4H 2 O).Separate solutions of calcium nitrate tetrahydrate (0.1 M) and magnesium nitrate tetrahydrate (0.1 M) were prepared.Both solutions were mixed, and sodium hydroxide (NaOH) pellets were added to maintain a pH 11.5.The mixture was stirred on a hotplate at 90°C, 120 g for 6 h.The formation of the Ca and Mg nanocomposite was confirmed by visually observing the color change of the solution.After 6 h, the solution was cooled to room temperature and centrifuged at 7,000g, 25°C for 5 min.The obtained residue was then washed several times with double-distilled water, dried in an oven at 80°C for 24 h, and ground using a mortar and pestle.The nanocomposite was calcined at 500°C for 3 h using a furnace tube to homogenize and remove impurities.X-ray diffraction (XRD) patterns and Fourier transform infrared (FT-IR) spectra were analyzed using PANalytical B•V.(Netherlands) and PerkinElmer, respectively.The surface morphology of the nanocomposite was examined by scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy (SEM-EDX) and transmission electron microscopy (TEM), as shown in Figure 1.

Seeds inoculation with B. pumilus
The isolated strain B. pumilus (KF859972) was collected from the Department of Microbiology, GC University, Faisalabad, and prepared following Zeng et al. (2020).Nutrient broth (250 mL) simmered on a rotary shaker (150 g, 37°C, 24 h), then collected after centrifugation (10,000g, 10 min).The supernatant was discarded, and the residue was washed with sterilized distilled water.Surface-sterilized seeds (hydrogen peroxide 10% H 2 O 2 ) were inoculated with bacteria using carboxymethyl cellulose (2%) on a rotary shaker at room temperature (90 g).A mixture of clay and peat moss (1:1 w/w) was used to coat the inoculated seeds.Both normal and inoculated seeds were planted in control and respective treatment conditions according to the treatment plan.

Experimental conditions
A pot experiment was carried out under natural environmental conditions (day-night temperature, 39/32°C, and humidity, 78 ± 4%) in 2022 at the Botanical Garden of Government College University Faisalabad, Pakistan.Treatments includes: Control; Cd 5 mg kg −1 , Cd 10 mg kg −1 , Microbes, Cd 5 mg kg −1 + Microbes, Cd 10 mg kg −1 + Microbes, (Ca + Mg) foliar, Cd 5 mg kg −1 + (Ca + Mg) foliar, Cd 10 mg kg −1 + (Ca + Mg) foliar, Microbes + (Ca + Mg) foliar, Cd 5 mg kg −1 + (Ca + Mg) foliar + Microbes, Cd 10 mg kg −1 + (Ca + Mg) foliar + Microbes.Pots were filled with sifted soil (5 kg pot −1 ) spiked with Cd (5 mg kg −1 and 10 mg kg −1 ) using cadmium chloride according to the treatment plan.A complete randomized design was used to conduct the experiment in triplicate.Rice seeds were soaked in water for 48 h, inoculated with B. pumilus before sowing, and grown in sieved and washed sand.After 20 days of germination, the rice plants were transplanted into pots, each containing four healthy plants.The recommended dose of NPK fertilizer was applied to prevent nutrient deficiency.The foliar Ca + Mg nanocomposite was applied after germination.A total of seven foliar applications were sprayed at one-and-a-half week intervals.

Measurement of photosynthetic pigments and gas exchange parameters
SPAD value was measured using an in situ SPAD meter.Photosynthetic pigments such as chlorophyll a and b, total chlorophyll, and carotenoids were determined spectrophotometrically in fresh rice leaves (Lichtenthaler, 1987).Acetone 85% (v/v ratio) was used to extract the samples to assess chlorophyll and carotenoid contents.Readings were taken using a spectrophotometer after extraction and centrifugation of samples.Gas exchange parameters (photosynthesis rate, transpiration rate, stomatal conductance, and water use efficiency) were measured during sunlight (12:00 a.m.) in leaves of rice plants using an infrared gas analyzer (IRGA).

Harvesting of plants
Plants were harvested and carefully separated into shoots, roots, leaves, and grains after 120 days of sowing.Growth parameters such as root and shoot lengths (cm), root fresh and dry weights (g), shoot fresh and dry weights (g), spike length, and number of grains were determined.Roots were washed with HCl (0.1%) and distilled water to remove metals.Root, shoot, and grain samples were oven-dried (72 h at 80°C) and crushed into small pieces for further analysis.

Measurement of oxidative stress and antioxidant enzymatic activity
Malondialdehyde (MDA) levels were assessed using the TBA (0.1%) method (Zhang and Kirkham, 1994;Abbas et al., 2017).Electrolyte leakage determination was achieved in two steps.Initial EC was noted after incubating samples for 2 h at 32°C.Second EC was measured for 20 min at 121°C following Dionisio-Sese and Tobita (1998).H 2 O 2 activity was analyzed as per Jana and Choudhuri (1982).Samples were crushed in phosphate buffer (PB 5 Mm and pH 6.5) and centrifuged for 20 min.Sulfuric acid (20%) was added after centrifugation and then centrifuged for 15 min.Absorbance was measured at a wavelength of 410 nm using a spectrophotometer.Superoxide radical (O 2
Phosphate buffer (PB 0.5, at pH 7.8) was used to homogenize the leaf and root samples for the determination of peroxidase (POD) and superoxide dismutase (SOD) contents (Zhang, 1992).Ascorbate peroxidase (APX) activity was estimated using the Nakano and Asada (1981) protocol, while CAT contents were assessed using the Aebi (1984) method.

Measurement of metabolites
Total soluble proteins (TSP) were measured by homogenizing 0.5 g of fresh leaves in potassium phosphate buffer (50 mM, pH 7.5) following Bradford (1976).Fresh leaves (0.5 g) were crushed in a potassium phosphate buffer solution (50 mM, pH 7.5).Pyridine and acid ninhydrin were used to titrate the supernatant to measure total free amino acids (TFAA) (Hamilton et al., 1943).Total soluble sugars (TSS) were analyzed by homogenizing 0.5 g of fresh leaves in an ethanol and ethanol mixture, and the extract was reacted with an anthrone reagent (Yemm and Willis, 1954).Phenolics were determined by triturating 0.5 g of fresh leaves in acetone, centrifuging at 10,000g for 10 min, and then reacting the supernatant with Folin and Ciocalteau's phenol reagent for determination (Wolfe et al., 2003).

Assessment of metal contents and macro and micronutrients
The protocol of Lwalaba et al. (2020) was adopted with slight modification.Briefly, samples were digested in a diacid mixture of HNO 3 :HClO 4 (4:1 v/v) at 140°C on a hotplate.Concentrations of elements such as Ca, Mg, Mn, Zn, Fe, K, and Cd were determined using ICP-MS (iCAP RQ, Thermo Scientific).

Statistical analysis
Data analysis was performed using SPSS version 16.0 (SPSS, Chicago, IL).One-way analysis of variance (ANOVA) was conducted, following the Tukey HSD test to assess significant differences among means.

Effect of Ca + Mg nanocomposite and B. pumilus on growth parameters
Foliar application of Ca + Mg nanocomposite and microbial inoculation showed positive effects on rice growth, physiology, and antioxidant contents under Cd (5 mg kg −1 , 10 mg kg −1 ) toxicity.The results showed that Cd (5 mg kg −1 ) reduced the length of roots and shoots by 20% and 30%, weight of fresh roots and shoots by 22% and 13%, weight of dry roots and shoots by 19% and 16%, number of tillers and grains by 38% and 19%, and spike length by 21% compared to the control treatment.Similarly, the roots and shoots length were reduced by 44% and 46%, weight of fresh roots and shoots by 42% and 32%, weight of dry roots and shoots by 36% and 37%, number of tillers and grains by 59% and 35%, and spike length by 41% significantly (p ≤0.05) inhibited with Cd (10 mg kg −1 ) compared with control treatment.Meanwhile, the B. pumilus significantly (p ≤0.05) increased the length of roots and shoots length by 23% and 22%, weight of fresh roots and shoots by 26% and 10%, wet of dry roots and shoots by 21% and 12%, number of tillers and grains by 20% and 19%, and spike length by 13% compared to the control.Moreover, B. pumilus inoculation enhanced the length of the roots and shoots by 3% and 12%, weight of fresh roots and shoots by 10% and 11%, weight of dry roots and shoots by 7% and 11%, number of tillers and grains by 46% and 16%, and spike length by 15% in Cd (5 mg kg −1 ) contamination compared without B. pumilus treatment.Under Cd (10 mg kg −1 ) contamination, B. pumilus improved the length of roots and shoots by 11% and 19%, weight of fresh roots and shoots by 15% and 19%, weight of dry roots and shoots by 9% and 25%, number of tillers and grains by 75% and 22%, and spike length by 24% compared without B. pumilus treatment.
Foliar application of Ca + Mg nanocomposite promoted the length of roots and shoots by 22 and 14%, weight of fresh roots and shoots by 23 and 10%, weight of dry roots and shoots by 18 and 15%, number of tillers and grains by 18 and 20%, and spike length by 26% compared to control treatment.Application of Ca + Mg nanocomposite boosted the length of roots and shoots by 17 and 15%, weight of fresh roots by 9%, weight of dry roots and shoots by 8 and 2%, number of grains by 4%, spike length by 4%, and decreased the weight of fresh shoots by 1% and number of tillers by 3% under Cd (5 mg kg −1 ) contamination compared to Cd (5 mg kg −1 ) + B. pumilus treatment.Similarly, Ca + Mg nanocomposite foliar application enhanced the length of roots and shoots by 21% and 18%, weight of fresh roots by 13%, dry roots and shoots weight 17% and 3%, number of grains by 3%, spike length by 4%, while decreasing the weight of fresh shoots by 2%, and no. of tillers by 7% under Cd (10 mg kg −1 ) contamination compared with Cd (10 mg kg −1 ) + B. pumilus treatment.
The highest increase was observed with the combined application of Ca + Mg foliar spray and B. pumilus.Significantly (p ≤0.05), the combined application of Ca + Mg and B. pumilus inoculation boosted length of roots and shoots by 14% and 8%, weight of fresh roots and shoots by 11% and 9%, weight of dry roots and shoots by 8% and 10%, number of tillers and grains by 21% and 16%, and spike length by 21% compared with B. pumilus treatment.While, the combined application of Ca + Mg and B. pumilus inoculation augmented the length of roots and shoots by 31% and 28%, weight of fresh roots and shoots by 17% and 9%, weight of dry roots and shoots by 15% and 14%, number of tillers and grains by 17% and 19%, and spike length by 23% with Cd (5 mg kg −1 ) contamination as compared to Cd (5 mg kg −1 ) + B. pumilus treatment.Similarly, the combined application of Ca + Mg and B. pumilus significantly (p ≤0.05) boosted the length of roots and shoots by 43% and 32%, weight of fresh roots and shoots by 27% and 16%, weight of dry roots and shoots by 24 and 14%, number of tillers and grains by 25% and 22%, and spike length by 29% under Cd (10 mg kg −1 ) as compared with Cd (10 mg kg −1 ) + B. pumilus treatment.

Effect of Ca + Mg nanocomposite and B. pumilus on photosynthetic pigments
The statistical analysis showed that the SPAD values, chlorophyll a, b, total chlorophyll, and carotenoid with Cd (5 mg kg −1 , 10 mg kg −1 ) toxicity decreased at a significant (p ≤0.05).

A B
(A) Alone and combined effect of Ca+Mg nanocomposite (25 mg L -1 ) and inoculation of Bacillus pumilus on total soluble proteins, total free amino acids, total soluble sugars and phenolics in roots and small letter showed the difference in significance at p ≤ 0.05 level with mean of three replications.(B) Alone and combined effect of Ca+Mg nanocomposite (25 mg L -1 ) and inoculation of Bacillus pumilus on total soluble proteins, total free amino acids, total soluble sugars and phenolics in leaves and small letter showed the difference in significance at p ≤ 0.05 level with mean of three replications.

Effect of nanocomposite (Ca + Mg) and B. pumilus on Cd uptake
Statistically, soil spiking with Cd (5 mg kg −1 , 10 mg kg −1 ) increased Cd levels in roots, shoots, and grains of rice over the control (Figure 4).Meanwhile, Ca + Mg nanocomposite and microbial inoculation significantly decreased the Cd uptake and toxicity with Cd (5 mg kg −1 , 10 mg kg −1 ) contamination.Meanwhile, B. pumilus inoculation at a significant (p ≤0.05) declined the uptake of Cd in roots, shoots, and grains by 61%, 55%, and 60%, compared to control.Moreover, B. pumilus inoculation reduced the uptake of Cd in roots, shoots, and grains by 35%, 47%, and 40% with Cd (5 mg kg −1 ) contamination, compared to without B. pumilus inoculation.Under Cd (10 mg kg −1 ) contamination, B. pumilus inoculation at a significant (p ≤0.05) lessened the uptake of Cd in roots, shoots, and grains by 26 % , 2 7 %, a n d 2 3% c o m p a r ed w i t h o u t B .p u m i l u s inoculation treatment.
Ca + Mg nanocomposite decreased the uptake of Cd in roots, shoots, and grains by 31%, 55%, and 60%, compared to the control.Meanwhile, the Ca + Mg nanocomposite augmented the uptake of Cd in roots, shoots, and grains by 18%, 24%, and 19% with Cd (5

Discussion
Several studies showed that the application of exogenic CaO and MgO NPs alleviates Cd toxicity in different crops (Li et al., 2022;Nazir et al., 2022).Similarly, B. pumilus strains increased plant growth by decreasing Cd contamination in soil (Shahzad et al., 2021;Maslennikova et al., 2023).However, there is a gap in research regarding the combined effect of Ca-Mg nanocomposite and microbes on plants and their mitigation mechanism.Therefore, the present study examined the combined effect of Ca-Mg and B. pumilus inoculation on physiology, photosynthetic pigments, oxidative stress, Cd uptake, and accumulation in rice.
After Cd uptake, the high excitation energy of thylakoid situated photosynthetic electron transport was produced, which ultimately promotes ROS synthesis (MDA, H 2 O 2, and EL) (Huihui et al., 2020).Cd also reduced the production of SOD, POD, CAT, and APX, which may hinder excessive scavenging of oxidative stress species.MgO NPs enhanced light absorption, photosynthetic function, photosystem II (PSII) efficacy, Fv/Fm, and the effective quantum yield of PSII photochemistry (FPSII).Magnesium also benefits net CO 2 absorption in several plant species and mitigates heavy metal stress (Tränkner et al., 2016;Samborska et al., 2018;Faizan et al., 2021).To minimize the toxic effects in heavy-metal-stressed plants, usually caused by the generation of reactive oxygen species (ROS), antioxidative protective mechanisms are activated, including the antioxidant enzymes CAT, POD, and SOD (Ahmad et al., 2018).Similarly, CaO NPs enhanced growth, antioxidative enzymes, and nutrient profile by inhibiting Cd uptake and toxicity in barley seedlings (Nazir et al., 2022).B. pumilus produces organic acids that bind C and solubilize phosphorus and other nutrients, which help plant growth (Sharma et al., 2013).The proposed mechanism of the Ca + Mg nanocomposite and B. pumilus strain is shown in Figure 5. Roots are exposed directly to the soil, and the Casparian strip might be an effective barrier to decrease Cd uptake and translocation due to the sole structure of the endothelial layer (Wu et al., 2018;Guo et al., 2021).Earlier findings discovered that Cd content in rice roots was 10 times higher than in upground plant parts, and a major portion of Cd accumulated in the cell wall, demonstrating that the root cell wall efficiently reduced Cd translocation to shoots and leaves (Liu et al., 2016;Yu et al., 2020).Cd concentration was significantly minimized with Ca, signifying an intervention between Ca 2+ and Cd 2+ ions.Cd is a nonessential and toxic element for rice plants, and Ca transporters enter the cells due to the chemical similarity (Tian et al., 2016;Ye et al., 2020).Through specific translocation channels, Ca 2+ ions impede the uptake and translocation of Cd 2+ ions by the roots, subsequently reducing the Cd concentration in plant parts (Kanu et al., 2019).Ca contents in the cytoplasm increased significantly, and signals were conveyed quickly among cells, allowing plants to mitigate Cd toxicity (Guo et al., 2018).Cd toxicity increased rice's amino groups, hydroxyl groups, cellulose, epoxide, and reactive oxygen species richness, causing structural damage to the plasma membrane and cell wall.At the same time, Alone and combined effect of Ca+Mg nanocomposite (25 mg L -1 ) and inoculation of Bacillus pumilus on Cd uptake and accumulation in roots, shoots, grains, and small letter showed the difference in significance at p ≤ 0.05 level with mean of three replications.
Ca minimized these unfavorable effects (Ye et al., 2020).Previous analyses indicated that Cd toxicity caused structural, chlorophyll alterations, primarily by replacing Mg 2+ ions, leading to the breakdown of chlorophyll fragments (Kanu et al., 2019).Similarly, Mg concentrations correlate negatively with Cd concentrations in rice leaves and shoots (Khaliq et al., 2019).
Cadmium is a non-essential element for plant growth with no biological function and inhibits the biomass of various plant species, including root and shoot length, dry weight, and SPAD values (Jia et al., 2020;Yang et al., 2020;Li et al., 2021).Similar results were found in the current study: Cd toxicity decreased rice plant growth parameters, including root and shoot length, root and shoot fresh and dry weight, number of tillers, and spike length, as shown in Table 2. Several studies have demonstrated that the exogenous application of NPs can alleviate Cd toxicity in wheat and rice, including Fe, Cu, TiO 2 , and ZnO NPs (Hussain et al., 2018;Noman et al., 2020;Irshad et al., 2021;Manzoor et al., 2021).It has been reported that CaO NPs could promote root development, growth rate, and seed yield, and supply Ca in soybean and peanut (Liu andLal, 2014, 2015).Ca and Mg are beneficial elements and enhance the plant growth.A previous study reported that the application of Ca and Mg improved growth by reducing Cd accumulation and translocation in rice (Li et al., 2022).Similarly, Mg input to chloroplast significantly enhanced the photosynthesis in rice (Li et al., 2020).Several microbes are well-known for detoxifying, transferring, and accumulating heavy metals (Pathania and Srivastava, 2020).A previous study reported that seed inoculation with B. pumilus increased maize growth by decreasing Cd toxicity (Shahzad et al., 2021).Fatemi et al. (2020) reported that the combined application of lead (Pb)-resistant microbes and silicon NPs improved the growth of coriander (Coriandrum sativum L.) under Pb stress.Previous studies showed that applying silica NPs with two microbial strains (Azotobacter chroococcum and Pseudomonas koreensis) improved the physiology and SPAD values of barely under saline stress (Alharbi et al., 2022).Our study also showed similar results: the combined application of Ca-Mg nanocomposite and B. pumilus inoculation minimizes Cd toxicity and increases plant growth factors (Table 3).
The literature confirmed that photosynthetic pigments are considered fundamental markers of heavy metals-induced oxidative stress (Rizwan et al., 2018).Previous studies demonstrated that CaO NPs increased chlorophyll content and gas exchange characteristics by reducing arsenic toxicity in barley.Ilyas et al. (2022) stated that seed inoculation with Bacillus sp.strains increased the chlorophyll contents in wheat plants.Moreover, chlorophyll content and gas exchange attributes significantly increased with the combined application of Ca + Mg and microbial inoculation.Previous studies also agreed that the combined application of ZnO NPs and Cr-resistant microbes increased chlorophyll content and gas exchange characteristics in wheat (Ahmad et al., 2022).The present study demonstrated similar findings: Ca + Mg enhanced chlorophyll and gas exchange characteristics in rice leaves under Cd contamination (Table 3).
Nano-fertilization has been considered a game-changer in addressing nutritional insecurity, especially in developing countries (Wang et al., 2020;Cao et al., 2022);.The application of CaO NPs decreased the Cd concentration in the roots, shoots, and grains of barley plants and increased the macro and micronutrients (Nazir et al., 2022).Similarly, gallic acid increased wheat growth and decreased the uptake of Cr in roots, shoots and grains under tannery wastewater stress (Nafees et al., 2024b).Previously, Ca and Mg reduced the Cd concentration in rice roots, shoots, and grains (Li et al., 2022).Similar results were found in our study: the combined application of foliar Ca-Mg and B. pumilus decreased the Cd concentration in rice roots, shoots, and grains (Figure 4) and increased the macro and micronutrients (Zn, Fe, Mg, Mn, K, and Ca) in shoots and grains (Table 1).Therefore, during phytoremediation, the foliar application of Ca + Mg nanocomposite and microbial (B.pumilus) inoculation will not only raise the market value of agricultural products but also improve the nutritional value of rice grains, which is of great significance in addressing hidden hunger.

Conclusion
The present study investigated how cadmium toxicity reduced plant growth, biomass, and gas exchange characteristics in rice.Additionally, Cd-stressed rice demonstrated increased MDA, H 2 O 2 , EL, and O 2 •-levels and higher Cd concentration.However, the application of Ca + Mg nanocomposite and B. pumilus individually modulated the antioxidant system of treated rice by improving the activity of CAT, SOD POD, and APX.Moreover, the combined application of Ca + Mg nanocomposite and B. pumilus further enhanced the growth and physiochemical features of rice seedlings grown under both standard and Cd-contaminated conditions.Further studies are required to understand the molecular mechanisms involved in Cd tolerance in different crops through the synergistic application of Ca + Mg nanocomposite and B. pumilus.
FIGURE 2 (A) Alone and combined effect of Ca+Mg nanocomposite (25 mg L -1 ) and inoculation of Bacillus pumilus on enzymatic antioxidants POD, SOD, APX and CAT and oxidants EL, MDA, H 2 O 2 in roots and small letter showed the difference in significance at p ≤ 0.05 level with mean of three replications.(B) Alone and combined effect of Ca+Mg nanocomposite (25 mg L -1 ) and inoculation of Bacillus pumilus on enzymatic antioxidants POD, SOD, APX and CAT and oxidants EL, MDA, H 2 O 2 in shoots and small letter showed the difference in significance at p ≤ 0.05 level with mean of three replications.

FIGURE 5
FIGURE 5Proposed mechanism of Ca + Mg nanocomposite and B. pumilus to mitigate Cd toxicity and enhance rice growth and yield.

TABLE 1
Macronutrients and micronutrients contents in shoots and grains under different treatments.

TABLE 2
Growth parameters under different treatments.

TABLE 3
Photosynthetic and gas exchange parameters under different treatments.
ROS production by reducing EL%, MDA, H 2 O 2 , and O 2 •concentrations and increased antioxidant enzyme activities by increasing POD, SOD, APX, and CAT concentrations in roots and leaves (Figures 2A, B). decreased