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Research Article
Application of vermiculite and Azospirillum brasilense, particularly in combination, improved wheat growth, grain yield, photosynthetic rate, stomatal conductance, and chlorophyll content while reducing electrolyte leakage and Cd accumulation in plant tissues under cadmium (Cd) stress.
Cadmium (Cd) contamination in agricultural soils poses a serious threat to crop productivity and human health. This study evaluated the potential of Vermiculite and the plant growth-promoting bacterium Azospirillum brasilense to alleviate Cd toxicity in wheat (Triticum aestivum L.). A pot experiment with three replications and nine treatments was conducted: T0 (Control), T1 (Cd 100 mg/kg), T2 (Cd 100 mg/kg + Vermiculite), T3 (Cd 100 mg/kg + A. brasilense), T4 (Cd 100 mg/kg + Vermiculite + A. brasilense), T5 (Cd 200 mg/kg), T6 (Cd 200 mg/kg + Vermiculite), T7 (Cd 200 mg/kg + A. brasilense), and T8 (Cd 200 mg/kg + Vermiculite + A. brasilense). Higher Cd levels caused more severe growth inhibition in wheat. However, treatments with vermiculite and A. brasilense significantly improved plant growth, reduced Cd accumulation in plant tissues, and enhanced antioxidant enzyme activities (SOD, CAT). Notably, the combined application of vermiculite and A. brasilense (T4 and T8) showed the most effective mitigation of Cd stress. At 100 mg/kg Cd (T4), wheat plants showed a 10.67% increase in height, 10.21% longer spike length, and 19.87% higher grain yield compared to Cd-only treatment. Under 200 mg/kg Cd stress (T8), the same combination led to a 44.83% increase in plant height, 17.63% spike length improvement, and a 44.83% yield increase relative to untreated Cd-stressed plants (T5). These results demonstrate that the synergistic use of Vermiculite and Azospirillum brasilense enhances wheat tolerance to Cd toxicity. Their combined application improves growth parameters, physiological traits, and yield under Cd-stressed conditions, providing an eco-friendly approach for remediating Cd-contaminated soils and sustaining wheat productivity.
Wheat (Triticum aestivum L.) is a vital staple food crop that provides a main energy source for millions of people1. As a primary food source, it is cultivated on vast expanses of agricultural land, particularly in temperate regions. Wheat's versatility, nutritional value, and adaptability to diverse environmental conditions make it a cornerstone of food security2. However, the yield potential of wheat is often compromised by different abiotic and biotic stress factors, leading to substantial losses in productivity. One of the pressing challenges facing wheat production today is soil contamination with heavy metals, particularly cadmium (Cd)2,3. Cadmium, a toxic heavy metal, can be introduced into agricultural soils through industrial activities, phosphate fertilizers, and irrigation with contaminated water. Once in the soil, Cd is readily absorbed by wheat plants, where it accumulates in various tissues, leading to oxidative stress, impaired physiological functions, and reduced growth3. The toxic effects of Cd on wheat not only diminish crop yield but also cause human health issues through the consumption of contaminated grain. Heavy metal toxicity and other factors, such as nutrient deficiencies, poor soil structure, drought stress, and suboptimal agronomic practices, contribute to the low yield of wheat4. Ever-present threat of climate change exacerbates these challenges, making it increasingly difficult to achieve sustainable wheat production5.
Vermiculite is a naturally occurring mineral that has gained significant attention in agriculture for its unique properties and benefits as a soil amendment. Composed primarily of hydrated magnesium-aluminum-iron silicate, Vermiculite is known for its ability to expand when heated, resulting in a lightweight, highly porous material6. This expanded form of Vermiculite exhibits excellent water retention and aeration capabilities, making it an ideal medium for improving soil structure and enhancing plant growth. In agricultural applications, Vermiculite serves as a critical component in soil mixes, particularly in areas with poor soil quality or in conditions that require enhanced water and nutrient management7. Vermiculite has the ability to retain water in the soil and maintain soil moisture under water stress. It is valuable in preventing waterlogging and ensuring a consistent supply of moisture to plant roots. Additionally, Vermiculite's high cation exchange capacity (CEC) enables it to provide the nutrients, such as potassium (K), calcium (Ca), and magnesium (Mg), that are available for the plants over time 8. Vermiculite's role in mitigating environmental stress factors, such as heavy metal contamination, is of particular interest in sustainable agriculture6. Due to its high surface area and affinity for heavy metals, Vermiculite can adsorb and immobilize toxic elements like cadmium (Cd) in the soil, reducing their bioavailability to plants. This not only protects crops from the detrimental negative effects of metal toxicity but also helps in maintaining soil health by removing the harmful substances6,9.
Azospirillum brasilense is a well-known plant growth-promoting rhizobacterium (PGPR) that has garnered substantial interest in agricultural research and practice due to its ability to enhance plant growth and resilience under various stress conditions10. Originally isolated from the rhizosphere of tropical grasses, A. brasilense is a free-living bacterium that colonizes the roots of a wide range of crops, including wheat (Triticum aestivum L.), where it establishes a beneficial relationship with the host plant11. The primary mechanisms by which A. brasilense promotes crop growth are by fixing atmospheric N2 (nitrogen), which it can readily absorb and use for the plant functions. This biological nitrogen fixation reduces synthetic fertilizers' requirements for plant growth, contributing to more sustainable agricultural practices1,11. A. brasilense is known for producing indole-3-acetic acid (IAA), which is a phytohormone and can improve root development; this root development can improve nutrient uptake. Beyond its growth-promoting effects, A. brasilense can also enhance plant tolerance to heavy metal toxicity12. In contaminated soils, A. brasilense can modulate the plant's physiological and biochemical responses, reducing the uptake and accumulation of metals like cadmium (Cd). The bacterium achieves this through several mechanisms, such as altering root exudation patterns, enhancing antioxidant enzyme activity, and producing extracellular polymeric substances that can bind to heavy metals, thereby reducing their availability to plants13. The potential of A. brasilense to mitigate Cd toxicity in wheat is particularly significant in the context of modern agriculture, where soil contamination with heavy metals poses a growing threat to crop productivity and food safety. By leveraging the natural capabilities of A. brasilense, farmers can improve the health and yield of wheat crops even in challenging environments, contributing to food security and environmental sustainability11,13,14.
The growing challenge of cadmium (Cd) contamination in agricultural soils presents a significant threat to the sustainability of crop production, particularly in staple crops like wheat3. Cd is a toxic heavy metal that not only hampers plant growth but can also accumulate in the edible parts of crops. As agricultural practices increasingly face the dual pressures of environmental pollution and the need for higher productivity, innovative and sustainable strategies are required to mitigate the impact of heavy metals like Cd on crop health15. The combined application of Vermiculite and Azospirillum brasilense offers a promising approach to addressing Cd toxicity in wheat7,9,14,16. Vermiculite, with its high cation exchange capacity and water retention properties, improves soil structure, enhances nutrient availability, and reduces the mobility of metals like Cd in the soil8. On the other hand, A. brasilense, a plant growth-promoting rhizobacterium, enhances plant tolerance to heavy metal toxicity by modulating root physiology, producing phytohormones, and reducing the bioavailability of toxic metals through various biochemical pathways14. The integration of vermiculite and A. brasilense in a single management strategy leverages the strengths of both components, creating a synergistic effect that is more effective than the use of either component alone17. Vermiculite's ability to immobilize Cd in the soil complements A. brasilense's role in enhancing plant defense mechanisms against Cd stress. Furthermore, Vermiculite can create a more conducive environment for A. brasilense to thrive by improving soil aeration and moisture levels, which are critical for the bacterium's survival and activity in the rhizosphere11,14. However, the combined effect of vermiculite and A. brasilense on wheat growth and physiological performance under Cd stress has not been systematically investigated.
The need for this combined application arises from the limitations of conventional approaches to managing Cd toxicity. Traditional methods, such as the use of chemical amendments, often provide only temporary relief and may have adverse environmental consequences. The combined use of vermiculite and A. brasilense can offer a more sustainable solution that not only mitigates Cd toxicity but can also enhance overall soil health and plant growth, leading to improved wheat yield, physiology, and quality. The combined application of vermiculite and A. brasilense on wheat growth and physiological performance under Cd stress has not been systematically investigated. To address this knowledge gap, the present pot experiment tested the following hypotheses: (H₁) Vermiculite application will reduce Cd accumulation in wheat tissues and improve growth and yield; (H₂) A. brasilense inoculation will enhance physiological performance (photosynthetic rate, stomatal conductance, chlorophyll content) and nutrient uptake; and (H₃) The combined application will produce greater improvements than either amendment alone, associated with higher bacterial root colonization. This study does not claim to provide direct field application guidelines, as those questions require field trials beyond the scope of this controlled experiment.
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All local, national, and international guidelines and legislation were adhered to for the use of plants in this study. The reagents and the equipment used are listed in the Table of Materials.
Experimental layout and material
A pot experiment was arranged at the rooftop of the agricultural engineering department building of Khwaja Fareed University of Engineering and Information Technology, Pakistan (28.42° N, 70.30° E) to investigate the role of vermiculite and Azospirillum brasilense to mitigate cadmium (Cd) toxicity in wheat crop during the winter season of 2023–2024. A completely randomized design (CRD) was used for this experiment, with three replications. An approved wheat variety of “Dilkash-20” was obtained from the Adoptive Research Farm, Rahim Yar Khan, which is suitable for the local region. A pot size 1 x 1 x 1 feet (30.48 cm × 30.48 cm × 30.48 cm) was used, having 12 kg of soil. Seed sterilization was performed before the experiment in the agronomy lab with 70% ethanol. Seeds were planted on 10th November, 2023. 8 wheat seeds were placed in each pot, and 4 plants were maintained at the tillering stage. Vermiculite was obtained from the local market and applied during the addition of the soil at a rate of 150 g/kg of soil. Azospirillum brasilense was obtained from the Government College University Lahore (GCUL), Lahore, Pakistan. Azospirillum brasilense was applied with the seed inoculation in wheat crop by the standard procedure as reported Zaheer et al.17. Experiment was consist of 9 treatments (T0 = Control (No soil amendment, No Cd toxicity), T1 = Cd toxicity 100 mg/kg of soil, T2 = Cd 100 mg/kg of soil + Vermiculite, T3 = Cd 100 mg/kg of soil + A. brasilense, T4 = Cd 100 mg/kg of soil + Vermiculite + A. brasilense, T5 = Cd toxicity 200 mg/kg of soil, T6 = Cd 200 mg/kg of soil + Vermiculite, T7 = Cd 200 mg/kg of soil + A. brasilense, T8 = Cd 200 mg/kg of soil + Vermiculite + A. brasilense). The average monthly temperature in °C and humidity in % at the experimental site are shown in Table 1. Before the start of the experiment, soil analysis was done, and soil properties are shown in Table 2. Irrigation was applied as per requirement at tillering, booting, anthesis, and grain filling stage, and fertilizers were also applied uniformly as per recommendations. Irrigation was applied manually with the use of a water jar and maintained 70% soil moisture in each pot, having no drought stress. Fertilizers were applied as per recommendations of the agriculture department (120-80-60 kg NPK ha-1). Fertilizers were calculated as per the size of each pot. Full doses of P and K and half a dose of N were applied at sowing time, and the remaining half dose of N was applied at the tillering stage. Destructive sampling for root and shoot length measurements (30 days after sowing) was performed on separate pots designated for early-stage harvest; these pots were not used for final yield measurements, so early destructive sampling did not affect later yield data. A schematic timeline of the experiment is as follows: Day 0 (soil preparation, Cd spiking, vermiculite mixing, seed inoculation, sowing), Day 10–30 (root sampling for qPCR), Day 30 (root and shoot length), Day 85 (anthesis: physiological measurements), and Day 120 (harvest: yield parameters). Plant samples were collected at three time points. At 10, 20, and 30 days after sowing (DAS), roots were collected from separate pots. At 30 DAS, separate pots were destructively harvested for root and shoot length. At 85 DAS (anthesis), physiological parameters were measured non-destructively. At 120 DAS, final yield parameters were recorded. Pots used for early destructive sampling (10–30 DAS) were not used for yield measurements.
Cadmium was applied as cadmium chloride (CdCl₂•2.5H₂O, analytical grade, ≥98% purity). The required amounts for target concentrations of 100 mg Cd kg⁻1 soil and 200 mg Cd kg⁻1 soil were calculated based on the dry weight of soil (12 kg per pot). CdCl₂ was dissolved in 500 mL of deionized water per pot and mixed thoroughly with the soil using a mechanical mixer for 15 min to ensure homogeneous distribution. After mixing, the spiked soil was placed in plastic-lined pots and allowed to equilibrate for 14 days at room temperature (25 ± 2 °C) with periodic mixing every 3 days to stabilize Cd speciation and allow aging. Soil moisture was maintained at 70% of field capacity during the equilibration period. After equilibration, representative soil samples (10 g per pot) were collected, air-dried, digested with HNO₃/HClO₄ (4:1, v/v), and analyzed for total Cd concentration using an atomic absorption spectrophotometer to verify actual Cd levels. Measured Cd concentrations were 98.4 ± 3.2 mg kg⁻1 for the 100 mg kg⁻1 treatment and 197.6 ± 5.1 mg kg⁻1 for the 200 mg kg⁻1 treatment, confirming that target levels were achieved. Physicochemical properties and heavy metal content of Vermiculite used in this study are shown in Table 3.
| Month | Temperature (°C) | Humidity (%) |
| November, 2023 | 28 | 56 |
| December, 2023 | 18 | 62 |
| January, 2024 | 17 | 63 |
| February, 2024 | 18 | 32 |
| March, 2024 | 21 | 25 |
| April, 2024 | 24 | 20 |
Table 1: Average temperature and humidity of the experimental site during 2023–2024.
| Parameters | Values (2023-24) |
| Organic matter | 0.56% |
| pH | 7.42 |
| EC | 227 µS/cm |
| T.S.S. | 0.54% |
| Available-P | 5.21 ppm |
| Available-K | 119 ppm |
| Saturation percentage | 34 |
| Soil separates: Sand, Silt, Clay (%) | 36%, 40 %, 24 % |
Table 2: Soil properties of the soil used for the experiment before the sowing of the crop.
| Property | Value | Metal | Concentration (mg kg⁻¹) |
| Grade | Horticultural grade | Cadmium (Cd) | < 0.01 (below detection limit) |
| Particle size | 2-4 mm | Lead (Pb) | 2.1 |
| pH (1:5 in H₂O) | 7.2 | Copper (Cu) | 1.8 |
| Cation exchange capacity (CEC) | 85.4 cmolc kg⁻¹ | Zinc (Zn) | 5.3 |
| Water-holding capacity (WHC) | 450% (w/w) | - | - |
| Bulk density | 0.12 g cm-3 | - | - |
| Application rate | 150 g kg⁻¹ soil (15% w/w) | - | - |
| Amount per pot (12 kg soil) | 1.8 kg | - | - |
Table 3: Physicochemical properties and heavy metal content of Vermiculite used in this study.
Studied traits
All plant growth-related parameters, such as Plant height, spike length, number of spikelets per spike, grains per spike, 1000-grain weight, and grain yield per plant, were recorded manually with the use of a meter rod and a weight balance by using a standard procedure. Biochemical analysis (determination of antioxidant enzyme activities) and physiological analysis (photosynthetic rate, stomatal conductance, chlorophyll content, electrolyte leakage (EL), crop growth rate (CGR), and leaf area index (LAI)) were taken at the anthesis stage after 85 days of sowing. Root and shoot length were noticed after 30 days of wheat plant growth, when plants were removed to maintain 4 plants in the pot after 20 days of sowing. An infrared gas analyzer (Cl-340) and chlorophyll meter (CL-1) were used to observe the photosynthetic rate, stomatal conductance, and chlorophyll contents, respectively. Electrolyte leakage (EL) was measured with the equation reported by Mussell and Staples18 having standard procedure reported by Zaheer et al.14. EL was calculated as: EL (%) = (Initial EC / Final EC) × 100, where Initial EC is the electrical conductivity measured after 24 h of incubation at 25 °C, and Final EC is the conductivity measured after autoclaving the samples at 121 °C for 20 min.
Dissolved organic nitrogen (DOC), dissolved organic carbon (DOC), and soil organic carbon (SOC) were measured with the standard procedure19. LAI and CGR were noticed as per the procedure20. N, P, and K uptake by the wheat plant was noticed, as reported in the procedure described by the previous report21.
Statistical analysis
All the data for this research were collected from three replications, compiled, and averaged. Data was analyzed statistically with the use of Statistic 8.1 and Microsoft Excel 2010. Data were analyzed using one-way ANOVA followed by Fisher's Least Significant Difference (LSD) test at p ≤ 0.05 for mean separation. Different letters showing with the data/results show the statistical difference between each treatment.
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Plant height (PH) was significantly affected by the soil amendments in all studied treatments (Table 4). The highest pH was noticed in T0 (199.44 cm), when there was no soil amendment and no Cd toxicity, followed by T4 (98.44 cm) when Cd toxicity was 100 mg/kg of soil with the application of vermiculite and A. brasilense. The lowest pH (82.37 cm) was noticed in T5 when there was 200 mg/kg of soil Cd toxicity. It was also observed that PH decreased with increasing Cd toxicity, and a significant i...
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Cd toxicity significantly reduced all measured growth and yield parameters in wheat22. This growth suppression can be attributed to Cd-induced oxidative stress, which disrupts cell division and elongation, impairs nutrient uptake, and reduces photosynthetic efficiency. Similarly, the number of spikelets per spike, grains per spike, grain weight, and yield per plant declined significantly under Cd stress. These findings align with previous studies highlighting the effect of Cd on plant physiologica...
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The authors declare that they have no competing interests. During the preparation of this manuscript, the author(s) used ChatGPT and Grammarly to improve the technical and English language of the paper. After using this tool/service, the author(s) reviewed and edited the content as needed and take full responsibility for the content of the publication.
The authors acknowledge the funding support provided by the Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Saudi Arabia, for this research (Grant No. KFU263053).
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| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| Atomic absorption spectrophotometer (AAS) | PerkinElmer, USA | AAnalyst 800 | Cd concentration verification in soil |
| Azospirillum brasilense | Government College University Lahore (GCUL), Pakistan | Strain available from GCUL culture collection | Seed inoculation |
| Cadmium chloride (CdCl2·2.5H2O) | Merck, Germany | CAS No. 7790-78-5 | Cd stress induction |
| Chlorophyll meter | Not specified | CL-1 | Chlorophyll content measurement |
| DNeasy PowerSoil Kit | QIAGEN, Hilden, Germany | Cat. No. 12888-100 | DNA extraction from roots |
| Excel 2010 | Microsoft, USA | Version 14.0 | Data compilation and basic statistics |
| Infrared gas analyzer (IRGA) | Not specified | CI-340 | Photosynthetic rate and stomatal conductance |
| nifH-specific primers | Custom synthesized | Sequences provided in text | qPCR amplification |
| Ninhydrin | Sigma-Aldrich, USA | CAS No. 485-47-2 | Proline content determination |
| QuantStudio 5 Real-Time PCR System | Applied Biosystems, USA | QuantStudio 5 | qPCR analysis |
| Spectrophotometer | Not specified | Not specified | Proline content measurement |
| Statistik 8.1 | Analytical Software, USA | Version 8.1 | Statistical analysis (ANOVA, LSD) |
| SYBR Green Master Mix | Applied Biosystems, USA | Cat. No. 4309155 | qPCR detection |
| Vermiculite (horticultural grade) | Local market, Rahim Yar Khan, Pakistan | Not applicable | Soil amendment |
| Wheat variety “Dilkash-20” | Adaptive Research Farm, Rahim Yar Khan, Pakistan | Local approved variety | Plant material |