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Addressing cadmium stress in rice with potassium-enriched biochar and Bacillus altitudinis rhizobacteria

BMC Plant Biology volume 24, Article number: 1084 (2024) Cite this article

Abstract

Cadmium (Cd) is a potentially harmful metal with significant biological toxicity that adversely affects plant growth and physiological metabolism. Excessive Cd exposure in plants leads to stunted plant growth owing to its negative impact on physiological functions such as photosynthesis, nutrient uptake, and water balance. Potassium-enriched biochar (KBC) and Bacillus altitudinis rhizobacteria (RB) can effectively overcome this problem. Potassium-enriched biochar (KBC) significantly enhances plant growth by improving the soil structure, encouraging water retention, and enhancing microbial activity as a slow-release nutrient. Rhizobacteria promote plant growth by improving root ion transport and nutrient availability while promoting soil health and water conservation through RB production. This study examined the effects of combining RB + KBC as an amendment to rice, both with and without Cd stress. Four treatments (control, KBC, RB, and RB + KBC) were applied using a completely randomized design (CRD) in four replications. The results showed that the combination of RB + KBC increased rice plant height (38.40%), shoot length (53.90%), and root length (12.49%) above the control under Cd stress. Additionally, there were notable improvements in chlorophyll a (15.31%), chlorophyll b (25.01%), and total chlorophyll (19.37%) compared to the control under Cd stress, which also showed the potential of RB + KBC treatment. Moreover, increased N, P, and K concentrations in the roots and shoots confirmed that RB + KBC could improve rice plant growth under Cd stress. Consequently, these findings suggest that RB + KBC is an effective amendment to alleviate Cd stress in rice. Farmers should use RB + KBC to achieve better rice growth under cadmium stress.

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Introduction

Cadmium (Cd) is a highly hazardous heavy metal that causes significant global soil contamination [1, 2]. Excessive Cd exposure in plants leads to stunted growth and development owing to its negative impact on physiological functions such as photosynthesis, food intake, and water balance [3]. It is a poisonous metal that quickly absorbs plant roots because of its water solubility and toxicity, altering plant structural and functional characteristics, preventing seed germination, and extending the roots [4]. Under Cd stress, plants produce highly reactive oxygen species, disrupt the plasma membrane system, and increase membrane fluidity, potentially affecting normal development [5].

As an essential macronutrient, potassium is necessary for human, animal, and plant health and improves soil fertility [6]. Its supplementation alleviates Cd stress, improves growth and photosynthesis, and reduces oxidative damage [7]. The defensive effects of K against Cd stress have also been attributed to increased nutrient uptake, decreased Cd translocation, improved antioxidant defense mechanisms, and better osmolyte production. These findings highlight the importance of optimizing K nutrition to reduce Cd toxicity in crops [8].

Biochar production is a promising method for carbon sequestration and sustainable agriculture [9,10,11]. Through controlled pyrolysis of specific feedstocks or adding potassium-bearing minerals during production, biochar can be enriched with potassium [12,13,14]. This enrichment can increase the carbon sequestration potential of biochar by up to 45% [15] and enhance its nutrient content, particularly that of potassium and phosphorus [16, 17]. K-enriched biochar acts as a slow-release fertilizer, improving soil fertility and reducing nutrient leaching [18]. The release of potassium from biochar is influenced by factors such as the pyrolysis temperature, soil type, and the physical form of biochar [16, 17].

Plant growth-promoting rhizobacteria (PGPR) have shown promise in mitigating cadmium (Cd) toxicity and enhancing phytoremediation. PGPR can reduce Cd phytotoxicity by improving plant growth, increasing root biomass, and enhancing Cd accumulation in plant tissues [19, 20]. Some PGPR strains, such as Enterobacter asburiae NC16, decrease Cd uptake by repressing iron uptake-associated pathways. PGPR can also immobilize Cd in the rhizosphere, reducing bioavailability [21]. PGPR inoculation in plants under Cd stress increases photosynthetic efficiency and secondary metabolism [22]. Adding rhizobacteria can manipulate soil pH and promote beneficial taxa [23]. Certain rhizobacteria can promote plant growth and Cd uptake in hyperaccumulators, potentially improving phytoremediation efficiency [24, 25].

Rice (Oryza sativa L.), a significant cereal crop, is a staple food for over half of the global population [26]. Global rice cultivation covers 167.13 million hectares, resulting in an annual rice production of 782 million tons [27]. Rice is an effective Cd accumulator because it absorbs and transports Cd from the soil into grains [28]. Reduced germination, biomass, root and shoot length, nutrient intake, and photosynthesis disruptions contribute to a slower rate of rice development and yield under cadmium stress [29]. The current knowledge on Cd stress in rice cultivation highlights the significant challenges Cd accumulation poses in rice plants, affecting crop yield and food safety [30]. Despite exploring various mitigation strategies, soil amendments [31], genetic approaches, and water management techniques, their effectiveness remains limited owing to variability in soil types, environmental conditions, and the complex interactions between Cd and other elements [30, 31]. These limitations underscore the need for more robust, adaptable, and sustainable strategies to mitigate Cd stress during rice cultivation.

While previous research has explored various methods to mitigate Cd stress in rice cultivation [32,33,34], the combined use of potassium-enriched biochar and rhizobacteria remains underexplored. Our study addresses this gap by investigating the synergistic effects of these two approaches, aiming to enhance rice plant resilience against cadmium stress and improve overall crop productivity. This study provides a novel and sustainable strategy for mitigating heavy metal contamination in rice fields. The primary objective of this study was to evaluate the effectiveness of K-enriched biochar and rhizobacteria in mitigating Cd stress in rice plants. It is hypothesized that applying potassium-enriched biochar to rhizobacteria might enhance rice plant growth, chlorophyll content, and photosynthetic efficiency and strengthen oxidative stress responses under cadmium stress.

Materials and methods

Experimental site

In 2022, the researcher conducted a pot experiment at their experimental site (30°09’41.6" N 71°36’38.0 " E = 19 Kassi Road, Kothiwala, Multan, Punjab, Pakistan), randomly sampling the soil to characterize its physicochemical attributes. The soil characteristics were as follows: Available phosphorus (P) = 6.21 µg/g dry soil [35]; soil organic matter (SOM) = 0.49% [36]; extractable potassium (K) = 122 µg/g dry soil [37]; ECe = 2.25 dS/m [38]; total nitrogen = 0.002% [39]; pH = 8.09 [40]; extractable sodium (Na), 111 µg/g dry soil [41]; extractable cadmium (Cd), 0.1 µg/g dry soil and clay loam in texture [41]. The irrigation conditions wre as follows: EC = 383 µS/cm; bicarbonates = 4.58 meq./L; Ca + Mg = 2.62 meq./L; carbonates = 0.01 meq./L; chloride = 0.01 meq./L; pH = 7.17 [42]; sodium = 103 mg/L. Following thinning, four seedlings were maintained per pot.

Potassium-enriched biochar (KBC) preparation and application

Initially, fruit waste was acquired from the local fruit market to serve as a carbon source for biochar synthesis. After drying, the biomass material was combined with potassium sulfate salt (Batch Number: WXBD9938V, Product Number: 223492, CAS Number: 7778–80–5, Brand: SIGALD) at a 2% level determined by the weight of the biomass. Subsequently, the biomass enriched in potassium was heated to 550 °C for 75 min while being pyrolyzed without oxygen. Following pyrolysis, KBC was cooled and stored in a suitable storage container. Biochar must be stored in a dry, well-ventilated space to preserve quality and prevent moisture absorption. For each treatment post, a KBC amendment was introduced at a rate of 0.70% (w/w) during soil preparation for pot filling. The soil was then manually mixed with KBC.

Characteristics of biochar

The biochar characterization was done following the standard methods provided as a reference for the studied attributes. The main characteristics of biochar were pHs [6.11] [43], ECe [6.21 dS/m] [38], volatile matter [13% w/w] [44], ash content [7% w/w] [44], fixed carbon [80% w/w] [44], extractable phosphorus [0.26%] [45], extractable cadmium [0.02 µg/g]. FTIR was performed to analyze functional groups. The small band at 855 cm− 1 was attributed to the vibrations of the saccharide structures of biochar. The band at 1022 indicated the symmetric and asymmetric C-O vibration of the C-O-C linkage. The absorption band at 1266 cm− 1 is assigned as C-N amine present in biochar. The band observed at 1460 cm− 1 may be ascribed to the bending vibrations of C-H alkanes, while another sharp band around 1522 cm− 1 frequency is due to the occurrence of N-H bending in amide II. The 1687 cm− 1 absorption band was identified as water molecules O-H stretching and vibration. The C ≡ C alkyne vibration is evident by an IR peak of about 2108 cm− 1. The band at 3119 cm− 1 is assigned to an N-H stretching vibrations and an O-H bond formed from important amides and starch in biochar (Fig. 1).

Fig. 1
figure 1

FTIR for the characterization of potassium-enriched biochar

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Isolation, incubation, and purification of PGPR isolates

Bacillus altitudinis was isolated by serial dilution of 1.0 grams of rhizosphere-derived homogenized soil between 10− 1 and 10− 7. A DF minimum salt medium was created with ACC (nitrogen source) having 10 µg/g Cd as contamination to aid in finding Bacillus altitudinis ability to produce ACC deaminase and their survival under Cd toxicity. The petri plates were incubated for 48 hours at 25°C. The characteristics of Bacillus altitudinis include shape [Rod], gram staining [+ ve], catalase [+ ve], exopolysaccharide [-ve], glucose utilization [+ ve], and methyl red [+ ve], ACC-deaminase activity [+ ve], indole acetic acid [+ ve], siderophore [+ ve] and chitinase [+ ve] [46,47,48,49]. Previously, this strain was identified [accession number = NR042337.1] based on comparing 16S rRNA partial sequencing with available data in GenBank using the BLAST algorithm. The primers 27F (5’-AGA GTT TGA TCM TGG CTC AG-3’ and 1492R (5’-TAC GGY TAC CTT GTT ACG ACT T-3’) were used for PCR amplification. The universal primers 785 F (5’-GGA TTA GAT ACC CTG GTA-3’) and 907R (5’-CCG TCA ATT CMT TTR AGT TT-3’) [50]. The Bacillus altitudinis successfully survived under this Cd toxicity.

Treatments plan

The treatments included control, KBC (0.70%), rhizobacteria (RB), and RB + KBC. All treatments were applied with no stress and Cd stress (20 mg Cd/kg soil toxicity). A completely randomized design (CRD) was used in the trial, with four replications for each treatment. After mixing biochar in the soil, it was incubated for 21 days so that biochar may become a homogenized part of the soil. The incubation conditions were 25 ± 5◦C with a humidity of 65%.

Cd contamination

The soil was collected from the experimental site at 30°09’41.6"N 71°36’38.0” E, and each pot with dimensions of 10-inch width and 12-inch depth was filled with 5 kg of Cd-contaminated soil. Cd contamination (6.20 mg/ kg soil) was induced by spiking the soil with Cd for 21 days. Cadmium nitrate tetrahydrate (642045 = product number, MKCT3996 = batch number, MFCD00149626 = MDL number, ALDRICH = brand, white = color) was used for this purpose.

Collecting, sterilization, and sowing of seeds

Rice seeds (Super Gold, fine-texture grains developed through traditional breeding methods) were purchased from a licensed seed dealer authorized by the government of Punjab, Pakistan. Sterilization was performed using a 5% sodium hypochlorite solution before seeding. After this, RB was then inoculated with peat, clay, and a sugar solution (10%). For 100 g of seeds, 10 ml inoculum (optical density 0.5), a 1:1 ratio of peat and clay mixture (10 g), and 10 ml of sugar solution were used. Sowing was performed when the seeds were air-dried. Flooding conditions in a pot were maintained by adding water to each pot, ensuring that the soil surface was submerged to a depth of 1 inch. Water levels were monitored daily and adjusted as necessary to maintain consistent flooding conditions throughout the growing period. Five seeds were planted in each pot. After germination, two healthy seedlings per pot were maintained by thinning procedure.

Fertilizer application

N, P, and K were applied at a 58:25:25 kg/acre ratio to meet the macronutrient requirements. Fertilizers such as urea, potassium sulfate, and single superphosphate were applied to fulfill the N, P, and K requirements.

Data collection

After harvesting, the morphological attributes of the plants were determined using an analytical grade balance. The dry weight of the samples was determined by heating in an oven at 70 °C for 48 h.

Chlorophyll content

To determine the chlorophyll content in the leaf samples, 20 ml of 80% acetone and a mortar and pestle were used to crush the fresh leaf sample (0.5 g). The mixture experienced 15-minute centrifugation at 3000 rpm to measure the absorbances at 645 nm and 663 nm [51].

Gas exchange attributes

The CI-340 Photosynthesis system was used to measure the gas exchange attributes. The measurements were performed on a sunny day from 10:30 AM to 11:30 AM, aligning with the time when light intensity reached saturation levels for photosynthesis [52].

N, P, and K shoot and root

Initially, for the analysis of nitrogen and sulfuric acid [53], phosphorus and potassium, a di-acid mixture [54] was used for sample digestion. A modified micro-Kjeldahl method was used to assess nitrogen content using the standard method [53]. Phosphorus content was determined at 420 nm using a spectrophotometer following the ammonium heptamolybdate and ammonium metavanadate yellow color method [53]. A flame photometer was used to analyze the potassium content of the digested samples. However, Cd was analyzed using an atomic absorption spectrophotometer [55].

Antioxidants

The superoxide dismutase (SOD) activity was assessed using nitro blue tetrazolium (NBT) at 560 nm [56]. Peroxidase (POD) activity was measured at 420 nm following standard protocol [57]. The decrease in absorbance due to H2O2 breakdown was determined at 240 nm as the CAT activity [58]. For ascorbate peroxidase activity, ascorbate oxidation in the presence of H2O2 was assessed at 290 nm (APX) [59]. A color complex was produced using thiobarbituric acid (TBA). The final absorbance was measured at 532 nm to determine malondialdehyde (MDA) [60]. Hydrogen peroxide (H2O2) was determined using a spectrophotometer at 390 nm following a standard protocol [61].

Leaf relative water content

The standard protocol [62] was adopted to evaluate relative water content.

$$\:\text{R}\text{W}\text{C}\:\left(\text{\%}\right)=\frac{\text{F}\text{W}-\text{D}\text{W}}{\text{T}\text{W}-\text{D}\text{W}}\times\:100$$

FW is the sample fresh weight, DW is the dry weight, and TW is the turgid weight.

Determination of nonenzymatic antioxidants

To measure glutathione (GSH), 5% sulfosalicylic acid (w/v) was used, and centrifugation was performed for 10 min at 12,000×g. The supernatant was mixed with 100 mM phosphate buffer (pH 7.0) and 5.5-dithiobis (2-nitrobenzoic acid). Finally, absorbance was measured at 412 nm using a spectrophotometer [63]. Trichloroacetic acid (10%) was used to determine AsA content. Centrifugation was performed for 10 min at 12,000×g. Absorbance was measured at 525 nm for the final assessment of AsA [64].

Total soluble proteins

The Biuret method [65] measured total soluble protein levels. Optical density was recorded using a UV-spectrophotometer at a frequency of 545 nm. Bovine serum albumin was used to create a protein standard curve to measure the total protein content.

Free proline

The free proline content was measured using the previously described procedure [66]. The components were extracted using sulfosalicylic acid, followed by the addition of ninhydrin and glacial acetic acid solutions. After adding toluene and heating the mixture to 100 °C, the absorbance of the toluene layer was measured at 520 nm.

Determination of total phenolic content

The Folin-Ciocalteu reagent method was employed alongside a standard curve for chlorogenic acid to quantify phenolic compounds in fresh shoots and root tissues. Absorbance readings were recorded at 740 nm to determine phenolic content [67].

Total flavonoid content

The total flavonoid content in the plant extract was calculated by combining 24µL of the extract (mg/mL) with 2824µL of NaNO2 (50 g/L) and incubating it for five minutes. Subsequently, 28 µL of AICI3 (100 g/L) was added, and the mixture was left to react for six minutes. The mixture was then mixed with 120 µL of 1 M NaOH, and the absorbance at 510 nm was measured immediately. TFC was calculated as mg/g fresh weight [68].

Statistical analysis

Standard statistical techniques were applied to analyze the data [69]. The study utilized OriginPro software, Excel 365, and MS Office. Tukey’s test was used to create paired comparison graphs at p ≤ 0.05, whereas Pearson correlation, convex hull cluster plots, and cluster plots were created using OriginPro software [70].

Results

Growth attributes

Under no stress, plant height increased by 9.24%, 4.21%, and 15.14% with KBC, RB, and RB + KBC, shoot length increased by 13.57%, 8.95%, and 20.77%, and root length increased by 14.64%, 5.47%, and 19.75% than the control. Adding KBC, RB, and RB + KBC treatments under Cd stress resulted in a 10.29%, 5.10%, and 38.40% increase in plant height, 44.05%, 25.88%, and 53.90% increase in shoot length, and 9.54%, 4.93%, and 12.49% increase in root length above the control, respectively (Fig. 2).

Fig. 2
figure 2

The effect of rhizobacteria (RB and potassium-enriched biochar (KBC) on plant height, shoot, and root length of rice grown with Cd stress and without stress. The Tukey test revealed significant differences (p < 0.05), with distinct letters representing the mean of four replicates

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Chlorophyll contents and RWC

The Addition of KBC, RB, and RB + KBC treatments with no stress resulted in an increase in chlorophyll a (9.02%, 4.97%, and 12.28%), chlorophyll b (11.49%, 4.39%, and 16.13%), total chlorophyll (9.70%, 4.81%, and 13.35%), and RWC (0.86%, 0.46%, and 1.35%) compared to the control. Under Cd stress, the applying KBC, RB and RB + KBC treatments resulted in a substantial increase in chlorophyll a (11.43%, 5.53%, and 15.31%), chlorophyll b (18.36%, 7.54%, and 25.01%), total chlorophyll (13.31%, 6.07%, and 19.37%) and RWC (1.11%, 0.56%, and 1.75%) compared to the control (Fig. 3).

Fig. 3
figure 3

The effect of rhizobacteria (RB and potassium-enriched biochar (KBC) on chlorophyll a, chlorophyll b, total chlorophyll, and RWC of rice grown with Cd stress and without stress. The Tukey test revealed significant differences (p < 0.05), with distinct letters representing the mean of four replicates

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MDA, H2O2, AsA, & GSH

MDA level decreased by 62.39%, 23.64%, and 89.37% under no stress; H2O2 level decreased by 2.23%, 1.22%, and 3.98%, respectively; AsA level decreased by 12.18%, 6.65%, and 23.34% and GSH level decreased by 18.17%, 10.57%, and 32.60% with KBC, RB, and RB + KBC treatments, respectively, to the control. Adding KBC, RB, and RB + KBC treatments under Cd stress, resulted in 30.64%, 14.88%, and 23.52% decreases in MDA, 4.78%, 2.02%, and 9.65% in H2O2, 10.31%, 5.23%, and 12.01% in AsA, and 10.13%, 5.66% and 14.18% decrease in GSH compared to the control (Fig. 4).

Fig. 4
figure 4

The effect of rhizobacteria (RB and potassium-enriched biochar (KBC) on MDA, H2O2, AsA, and GSH of rice grown with Cd stress and without stress. The Tukey test revealed significant differences (p < 0.05), with distinct letters representing the mean of four replicates

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Total soluble protein, proline, total phenolics, and total flavonoids

Applying KBC, RB, and RB + KBC treatment resulted in a 35.63%, 17.93%, and 63.33% increase in total soluble protein under no stress, proline content decreased by 32.07%, 15.38%, and 51.38%, total phenolics decreased by 12.29%, 5.20%, and 16.35%, and total flavonoids decreased by 9.97%, 5.27%, and 16.46% from the control. Under Cd stress, the application of KBC, RB, and RB + KBC treatments increased total soluble protein (16.31%, 6.85%, and 27.55%, respectively) and caused a decrease in proline (21.52%, 7.36%, and 16.28%, respectively), total phenolics (10.13%, 4.34%, and 13.76%, respectively), and total flavonoids (9.35%, 5.05%, and 12.49%, respectively) above the control (Fig. 5).

Fig. 5
figure 5

The effect of rhizobacteria (RB and potassium-enriched biochar (KBC) on total soluble protein, proline, total phenolics, and total flavonoids of rice grown with Cd stress and without stress. The Tukey test revealed significant differences (p < 0.05), with distinct letters representing the mean of four replicates

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POD, SOD, CAT, & APx

The POD level decreased by 25.92%, 12.06%, and 35.27% under no stress, SOD level decreased by 25.54%, 10.19%, and 34.28%, CAT level decreased by 34.20%, 15.17%, and 64.18%, and 17.89%, 7.55%, and 29.08% decrease in APX with KBC, RB, and RB + KBC compared with the control. The KBC, RB, and RB + KBC treatment showed 13.16%, 4.60%, and 19.10% decreases in POD, 9.03%, 3.36%, and 12.41% in SOD, 21.94%, 7.85%, and 21.15% in CAT, and 11.93%, 5.63%, and 15.53% in APX, respectively, under Cd stress compared with the control (Fig. 6).

Fig. 6
figure 6

The effect of rhizobacteria (RB and potassium-enriched biochar (KBC) on POD, SOD, CAT, and APX of rice grown with Cd stress and without stress. The Tukey test revealed significant differences (p < 0.05), with distinct letters representing the mean of four replicates

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Gass exchange attributes

Applying KBC, RB, and RB + KBC treatments under no stress significantly increased the photosynthetic rate (19.93%, 7.86%, and 30.22%, respectively), transpiration rate (19.45%, 11.39%, and 31.88%, respectively), stomatal conductance (27.12%, 13.56%, and 40.68%, respectively), and Intercellular CO2 concentration (4.68%, 2.17%, and 7.15%, respectively) were measured more than control. Under Cd stress, the addition of KBC, RB, and RB + KBC treatments showed a significant improvement in photosynthetic rate (18.33%, 9.70%, and 32.08%, respectively), transpiration rate (35.40%, 12.98%, and 69.28%, respectively), stomatal conductance (57.89%, 29.82%, and 85.96%, respectively), and intercellular CO2 concentration (3.53%, 1.28%, and 5.57%, respectively) over the control respectively (Fig. 7).

Fig. 7
figure 7

The effect of rhizobacteria (RB and potassium-enriched biochar (KBC) on photosynthetic rate, transpiration rate, stomatal conductance, and intracellular CO2 concentration of rice grown with Cd stress and without stress. The Tukey test revealed significant differences (p < 0.05), with distinct letters representing the mean of four replicates

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Nutrient analysis

Under no stress, the KBC, RB, and RB + KBC treatments significantly enhanced shoot N (6.09%, 3.39%, and 7.95%, respectively), shoot P (4.78%, 2.63%, and 7.81%, respectively), and shoot K (5.12%, 2.69%, and 7.05%, respectively) than the control. The increase in shoot N (7.47%, 4.38%, and 12.74%, respectively), shoot P (7.42%, 3.71%, and 12.75%, respectively), and shoot K (8.84%, 5.14%, and 13.75%, respectively) were observed in the control with KBC, RB, and RB + KBC treatments under Cd stress. Treatment KBC, RB, and RB + KBC showed an increase in root N (4.94%, 2.41%, and 7.71%, respectively), root P (9.56%, 4.07%, and 13.78%, respectively), and root K (14.79%, 7.71%, and 22.92%, respectively) over the control under no stress. A significant rise in root N (9.48%, 4.67%, and 14.57%, respectively), root P (14.21%, 7.47%, and 18.82%, respectively), and root K (21.52%, 7.27%, and 34.24%, respectively) was observed above the control with KBC, RB, and RB + KBC treatments under Cd stress (Fig. 8).

Fig. 8
figure 8

The effect of rhizobacteria (RB and potassium-enriched biochar (KBC) on shoot and root N, P & K of rice grown with Cd stress and without stress. The Tukey test revealed significant differences (p < 0.05), with distinct letters representing the mean of four replicates

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Cd in shoot and root

In comparison to the control, applying KBC, RB, and RB + KBC resulted in 21.79%, 10.48%, and 28.12% decreases in Cd in the shoot, and 20.53%, 11.83%, and 36.47% decrease in Cd in root under no stress. Adding KBC, RB, and RB + KBC treatment under Cd stress led to a 7.70%, 4.04%, and 12.47%, respectively, decrease in Cd in a shoot, and 23.03%, 6.69%, and 31.15%, respectively decrease in Cd in root over the control (Fig. 9 and 10).

Fig. 9
figure 9

The effect of rhizobacteria (RB and potassium-enriched biochar (KBC) on Cd in shoot and Cd in the root of rice grown with Cd stress and without stress. The Tukey test revealed significant differences (p < 0.05), with distinct letters representing the mean of four replicates

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Fig. 10
figure 10

In the current experiment, rice plants cultivated under no Cd and Cd stress were amended with potassium-enriched biochar (KBC) and rhizobacteria (RB)

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Convex hull and hierarchical cluster analysis

The Control group had positive and negative PC1 scores, ranging from − 9.11772 to 2.34916, and a relatively small variation in PC2 scores, ranging from − 0.46586 to 0.79455. KBC treatment also had a wide range of PC1 scores, from − 4.41606 to 6.66094, but the PC2 scores remained consistently negative, with values between − 0.35706 and − 0.10201. The RB treatment showed a similar pattern, with PC1 scores ranging from − 6.97546 to 4.53547 and PC2 scores varying from − 0.32763 to 0.34851. The combined RB + KBC treatment had PC1 scores ranging from − 1.88275 to 9.21746 and PC2 scores ranging from − 1.15183 to 0.77376 (Fig. 11A).

Much of the no-stress scores cluster on the positive side of PC1, ranging from 0.35062 to 9.21746, indicating a strong positive relationship between the measured variables in unstressed plants. In contrast, the Cd stress scores were clustered in the negative PC1 range from − 9.11772 to -0.15971, suggesting a significant shift in plant response under cadmium stress conditions. For plants under no stress, the PC1 scores increase progressively, showing a trend of growth and physiological stability. This group remained consistently on the right side of the PC1 axis, with small variations in PC2, generally maintaining values between − 1.15183 and 0.79455. This indicates a minimal deviation in the secondary principal component.

Conversely, the Cd Stress group presented consistently negative PC1 scores, indicating decreased physiological function due to Cd exposure. The PC1 scores range from − 9.11772 to -0.15971, with little variation in the PC2, which stayed between − 0.46586 and 0.77376. This clustering of Cd-stressed plants in the negative region of the PC1 highlights the substantial impact of Cd on the primary physiological parameters measured in this study (Fig. 10B).

At a very high similarity level (0.07852), proline and catalase (CAT) were grouped, indicating their strong association, possibly reflecting their role in stress response. Likewise, chlorophyll a and total chlorophyll formed another tight cluster at 0.10242, showing their close biochemical relationship, as they both reflect photosynthetic capacity. Another important cluster was observed between relative water content (RWC) and shoot phosphorus (Shoot P), with a similarity of 0.12297. This suggests that the shoot’s water status and phosphorus levels are interrelated, potentially indicating their combined influence on plant health and growth. Malondialdehyde (MDA) and ascorbate peroxidase (APX) cluster together at a similarity level of 0.14234, highlighting the role in the oxidative stress response, with MDA as a marker of lipid peroxidation and APX as a key antioxidant enzyme. Notably, shoot potassium (Shoot K) and root nitrogen (Root N) are clustered at a similarity level of 0.19325, reflecting a potential connection between nutrient distribution and uptake in different plant organs under stressful conditions.

Similarly, stomatal conductance and root potassium were grouped as 0.24621, suggesting a link between gas exchange and nutrient status. Total phenolic content clustered with other variables, such as total flavonoids and root phosphorus, with a similarity value of 0.3691, reflecting the coordinated role of these antioxidant compounds in plant stress tolerance. Plant height and shoot length exhibit less similarity at higher dissimilarity levels, clustering at 1.36824 and 1.04405, respectively, indicating their relatively independent behavior in overall plant growth. The most dissimilar variables are hydrogen peroxide (H2O2) and superoxide dismutase (SOD), with similarity levels of 1.86712 and 1.23482, respectively. These two components are important markers of oxidative stress and enzymatic defense mechanisms (Fig. 11C).

Fig. 11
figure 11

Cluster plot convex hull for treatment (A), cadmium (Cd) levels (B), and hierarchical cluster plot (C) for studied attributes

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Pearson correlation analysis

Plant height was strongly positively correlated with shoot length (r = 0.94991), root length (r = 0.93382), chlorophyll a (r = 0.96709), chlorophyll b (r = 0.96324), total chlorophyll (r = 0.97046), and relative water content (RWC) (r = 0.98037), indicating that increases in these factors were associated with greater plant height. Chlorophyll a, b, and total chlorophyll exhibited high correlations with each other, with the highest being between total chlorophyll and chlorophyll a (r = 0.99797), which emphasizes the close relationship between these pigments and the plant’s photosynthetic efficiency. There was a strong negative correlation between malondialdehyde (MDA) and parameters such as total chlorophyll (r = -0.99756), RWC (r = -0.99348), and shoot length (r = -0.98601), suggesting that as oxidative stress increases (indicated by MDA), these growth parameters decrease. Antioxidant activities, such as ascorbic acid (AsA) and glutathione (GSH), also showed negative correlations with MDA (r = -0.99625 for AsA and r = -0.9903 for GSH), implying that these antioxidants counteract oxidative stress. Moreover, heavy metal accumulation (Cd content in shoot and root) is negatively correlated with parameters like plant height (r = -0.95683 in shoot and r = -0.96848 in root), chlorophyll (r = -0.98679 for Cd in shoot and r = -0.99025 for Cd in root), and antioxidant enzymes such as catalase (r = -0.97915 for Cd in shoot and r = -0.9716 for Cd in root), demonstrating the inhibitory effects of Cd on plant growth and physiological function. Photosynthetic rate (r = 0.94399) and transpiration rate (r = 0.98707) were positively correlated with plant height, shoot length, and chlorophyll content, indicating that these processes are crucial for plant development. Negative correlations between the photosynthetic rate, MDA (r = -0.97274), and H2O2 (r = -0.93051) suggest that oxidative stress adversely affects the photosynthetic machinery. Nutrient uptake (N, P, and K) in shoots and roots was also positively correlated with plant growth metrics and photosynthetic pigments, emphasizing their roles in overall plant health. However, a high negative correlation between nutrient uptake and MDA (ranging from − 0.99225 to -0.99424) highlights the impact of stress on nutrient efficiency (Fig. 12).

Fig. 12
figure 12

Pearson correlation analysis for the studied attributes

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Discussion

Higher uptake of Cd in plants causes an imbalance in mineral nutrition, which inhibits physiological processes, such as photosynthesis [71, 72]. Such inhibition of photosynthesis results in poor plant growth, yield, and grain quality [73]. It also caused oxidative damage owing to changes in the metal transporter gen, affecting iron homeostasis [74]. Furthermore, negative changes in gas exchange attributes are key to minimizing plant growth under Cd toxicity [75]. The current study also noted similar results where plants showed poor growth, chlorophyll contents, and nutrients in the control treatment under Cd toxicity [76].

Plant growth parameters such as height, shoot length, and root length increased in all treatments under Cd stress [77]. This can be attributed to the ability of biochar to enhance soil structure, nutrient availability, and water retention, providing essential elements such as potassium [78]. Biochar also creates a favorable environment for beneficial microbial communities, supporting nutrient uptake and root growth [79]. Inoculation with rhizobacteria promotes plant growth by producing growth hormones and solubilizing phosphates, which are essential for plant development [80]. The combined application of RB and KBC yielded the most substantial increase in plant height and shoot length, likely due to improved root colonization, nutrient uptake, and stress mitigation. The inoculation of rhizobacteria, which is beneficial for plant growth, is an established strategy [81, 82]. These rhizobacteria modulate the hormonal balance, that is, ethylene, by decreasing the uptake of Cd in plants [82, 83]. It also facilitates cellular detoxification due to immobilization by secretion of abscisic acid (ABA), downregulating IRT1-mediated Cd uptake in plant roots [84]. The application of rhizobacteria as a treatment also improves plants’ antioxidant defense systems, minimizing oxidative damage caused by Cd toxicity in plants [82]. Our results in the current study also align with the above arguments, where RB decreased the Cd concentration and improved the antioxidant and nutrient uptake in plants under Cd stress.

RB + KBC treatment also led to reduced levels of antioxidant enzymes such as peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX), signaling a decrease in oxidative stress. This reduction is likely due to the capacity of KBC to adsorb excess reactive oxygen species (ROS) and RB to bolster the plant’s antioxidant defenses [85]. Lower levels of oxidative stress markers, such as malondialdehyde (MDA) and hydrogen peroxide (H2O2), along with reduced ascorbic acid (AsA) and glutathione (GSH) levels, further confirm that the treatments help alleviate oxidative damage and maintain cellular health. Additionally, the treatments resulted in lower levels of stress-related biochemical markers such as total soluble protein, proline, total phenolics, and total flavonoids, indicating that the plants experienced less stress overall. The reductions in these markers suggest that the treatments help maintain plant health and reduce the need for stress-induced protective compounds.

Chlorophyll content, a key marker of photosynthetic activity, also improved across treatments, particularly under stress conditions [86]. Potassium plays a crucial role in regulating photosynthesis, and KBC, which is rich in potassium, helps to maintain high chlorophyll levels [87]. Rhizobacteria protect chlorophyll from oxidative damage caused by stress and enhance the plant’s defense mechanisms [88]. This was reflected in the increase in chlorophyll content, especially in the RB + KBC treatment, which also improved the relative water content, suggesting better water uptake and retention. The role of potassium in stomatal regulation and rhizobacteria’s promotion of nutrient availability contributed to better gas exchange and water transport, which is crucial for sustaining photosynthesis under stress [89].

In recent studies, many scientists have observed that biochar application minimizes plant stress [87, 90]. It can decrease the uptake of Cd in plants owing to the formation of stable complexes with (oxy) hydroxides, carbonates, and organically bound phases in biochar [91, 92]. Better uptake of water and nutrients due to adding biochar plays an imperative role in improving plant growth when cultivated under Cd stress [93]. The use of biochar was the novelty aspect of the current study. This enrichment mainly focused on the ability of K to mitigate Cd stress in plants. Potassium enhances the enzymatic activities of antioxidants, which are key factors in decreasing oxidative stress [94, 95]. It also increases the synthesis of secondary metabolites and modifies the composition of the root cell wall, which enhances Cd retention and decreases Cd uptake in plants [96, 97]. K is also vital in chlorophyll synthesis, carbon fixation, and the electron transport chain [98, 99].

Conclusion

In conclusion, Bacillus altitudinis RB + 0.70%KBC has shown considerable potential to enhance rice growth under cadmium (Cd) stress, offering a promising strategy for sustainable agriculture. This treatment improves growth and strengthens rice resilience by modulating antioxidant levels, boosting chlorophyll content, and minimizing the harmful effects of cadmium. Given these positive findings, further research is suggested at the field level to validate and optimize Bacillus altitudinis RB + 0.70%KBC’s effectiveness in diverse agricultural environments. These insights are particularly valuable for growers in cadmium-affected regions, as Bacillus altitudinis RB + 0.70%KBC could significantly mitigate heavy metal toxicity in crops, thus supporting food safety and environmental health. We encourage researchers and growers alike to explore and advance the use of Bacillus altitudinis RB + 0.70%KBC as part of an integrated approach to sustainable crop production.

Data availability

All data generated or analyzed during this study are included in this published article.

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Acknowledgements

This project was supported by Researchers Supporting Project number (RSP2024R98), King Saud University, Riyadh, Saudi Arabia.

Funding

This project was supported by National Natural Science Foundation of China (No. 42167014), Natural Science Foundation of Jiangxi Province (No.20202BAB213016), Educational Commission of Jiangxi Province (No. GJJ180592). This project was supported by Researchers Supporting Project number (RSP2024R98), King Saud University, Riyadh, Saudi Arabia.

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Authors and Affiliations

  1. School of Life Science, Jinggangshan University, Ji’an, Jiangxi, 343009, China

    Yonghui Liao

  2. College of Life and Health Science, Anhui Science and Technology University, Fengyang, Anhui, 233100, China

    Shoucheng Huang

  3. Department of Environmental Sciences, Woman University Multan, Multan, Punjab, Pakistan

    Misbah Hareem

  4. Department of Soil and Environmental Sciences, Muhammad Nawaz Sharif University of Agriculture Multan, Multan, Punjab, Pakistan

    Muhammad Baqir Hussain

  5. Department of Botany and Microbiology, College of Science, King Saud University, P. O. Box.2455, Riyadh, 11451, Saudi Arabia

    Abdullah A. Alarfaj & Sulaiman Ali Alharbi

  6. Zoology Department, College of Science, King Saud University, Riyadh, 11451, Saudi Arabia

    Saleh Alfarraj

Authors
  1. Yonghui Liao
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  2. Shoucheng Huang
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  3. Misbah Hareem
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  4. Muhammad Baqir Hussain
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  5. Abdullah A. Alarfaj
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  6. Sulaiman Ali Alharbi
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  7. Saleh Alfarraj
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Contributions

Conceptualization; M.H.; Y.L.; S.H.; Conducted experiment; M.H.; Formal analysis; M.H.; Y.L.; S.H.; Methodology; M.H.; M.B.H.; Writing—original draft; M.H.; A.A.A.; S.A.A.; Writing—review & editing; M.H. S.A.; M.B.H.

Corresponding author

Correspondence to Misbah Hareem.

Ethics declarations

Ethics approval and consent to participate

I declare that manuscript reporting studies do not involve human participants, data, or tissue. So, it is not applicable. Experimental research and field studies on plants (cultivated or wild), including the collection of plant material, must comply with relevant institutional, national, and international guidelines and legislation I confirmed that all methods were performed according to the relevant guidelines/regulations/legislation. The seeds were purchased from a local certified seed dealer of the Government of Punjab, Pakistan.

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Not applicable.

Competing interests

The authors declare no competing interests.

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Liao, Y., Huang, S., Hareem, M. et al. Addressing cadmium stress in rice with potassium-enriched biochar and Bacillus altitudinis rhizobacteria. BMC Plant Biol 24, 1084 (2024). https://doi.org/10.1186/s12870-024-05793-z

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Keywords

BMC Plant Biology

ISSN: 1471-2229

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