elocation-id: elocation-id: e3742
This study explores the role of Bradyrhizobium sp. ( Lotus ) in enhancing the biochemical resilience of Lotus ornithopodioïdes during germination under metal stress conditions. Specifically, the effects of cadmium (Cd) and copper (Cu) on germination and various biochemical parameters were assessed. Seeds were inoculated with Bradyrhizobium sp. ( Lotus ) and subjected to different concentrations of trace metals. Key biochemical parameters, including proline, amino acids, total soluble sugars, α-amylase activity, and DNA content, were analyzed. The results demonstrated that inoculation with Bradyrhizobium sp. ( Lotus ) significantly mitigated the adverse effects of metal stress, leading to improved germination rates and enhanced biochemical responses. Notably, proline and amino acid levels were reduced, while α-amylase activity and DNA content increased in inoculated seeds compared to non-inoculated controls. These findings suggest that Bradyrhizobium sp. ( Lotus ) plays a vital role in promoting resilience in Lotus ornithopodioïdes under metal stress, highlighting its potential for use in bioremediation and sustainable agricultural practices.
biochemistry, cadmium, copper, germination, metal stress, PGPR, trefoil.
Heavy metal contamination of agricultural soils is a growing global concern, posing significant challenges to crop production and food security ( Rizwan et al ., 2016 ). Toxic trace metals such as cadmium (Cd) and copper (Cu) can severely impair seed germination, plant growth and overall crop yield ( Hossain et al ., 2012 ).
The rising presence of heavy and trace metals in agricultural systems calls for sustainable, eco-friendly strategies to reduce plant stress. One effective approach is the use of plant-microbiome interactions, particularly plant growth-promoting rhizobacteria (PGPR) ( Etesami and Maheshwari, 2018 ).
Bradyrhizobium species are well-known PGPR that enhance growth, nutrient uptake, and stress tolerance in legumes, including mitigating metal stress ( Ahemad and Kibret, 2014 ; Shameer and Prasad, 2018 ). However, the mechanisms by which Bradyrhizobium sp. ( Lotus ) modulates the biochemical responses of Lotus ornithopodioïdes L. during germination under metal stress remain poorly understood.
The research focuses on evaluating the impact of Bradyrhizobium sp. ( Lotus ) inoculation on key biochemical parameters, including proline, amino acids, total soluble sugars, α-amylase activity and DNA content, under varying concentrations of trace metals.
Bradyrhizobium sp. ( Lotus ) described by Degaichia et al . (2024) was used at a concentration of 108CFU ml-1.
The germination of the Lotus ornithopodioïdes L. seeds and the conduct of the test is carried out according to the work protocol of Degaichia et al . (2025) .
Seed inoculation with Bradyrhizobium sp. ( Lotus ) was performed following Silini et al . (2016) . Seeds were germinated in Petri dishes (20 seeds per dish) on filter paper moistened with distilled water (control) or CdCl2and CuCl22H2O solutions ( Degaichia et al ., 2025 ) ( Table 1 ). Germination was conducted in the dark at 25 °C.
| Cu(II) | Cd(II) | Cu(II):Cd(II) |
|---|---|---|
| 1.5 | 2 | 1.5:2 …. I |
| 2 | 3.5 | 2: 3.5…. II |
| 3 | 6 | 3:6 …. III |
After the third day of incubation, the analysis of the biochemical parameters was carried out ( Degaichia et al ., 2025 ).
The proline content was determined by the method developed by Singh et al . (1973) . The absorbance was read at 520 nm. The results are reported on a proline standard curve.
The samples were stored below -15 °C before analysis. The extraction was carried out according to the method described by Naidu et al . (1998) . The results were expressed in µmoles of equivalents leucine per gram of fresh matter (µM equiv leucine g-1FM).
The extraction of sugars is carried out according to Babu et al . (2002) . The determination of total soluble sugars was carried out using the Dubois method ( Dubois et al ., 1956 ). The results of the optical densities were reported on the standard curve of soluble sugars (expressed as glucose).
Alpha-amylase was measured according to the method of Xiao et al . (2007) . The results were expressed in enzymatic units per milliliter (U ml-1), calculated according to the following formula:
Where: A580T= absorbance of starch without addition of the enzymatic extract; A580E= absorbance of starch after addition of the enzymatic extract; A580ET= absorbance for 1 mg of starch derived from the standard curve; T= incubation time in minutes; Vext= volume of the added enzymatic extract in milliliters.
DNA was measured according to the technique used by Burton (1956) . The optical density reading of the samples was carried out at 600 nm (OD600). The DNA content is determined according to the formula:
In all tables, Conc denotes TM concentration; N ino and Ino indicate non-inoculated and inoculated seeds with Bradyrhizobium sp. ( Lotus ), respectively. Values represent means. Different uppercase letters within rows and lowercase letters within columns indicate significant differences according to student’s t-test and Tukey’s test, respectively (p≤ 0.05).
In the control, 0.97 µg g-1FM of Proline content is recorded, this value increases significantly with the increase in the Cd(II) concentration and reaches 1.41 µg g-1FM at 6 g L-1of Cd(II). In a copper medium, the Proline content is positively correlated with the concentration, where a maximum of 1.55 µg g-1FM is recorded for the concentration 3 g L-1. The mixture of the two elements also generates significant increases compared to the control ( Table 2 ).
Bacterial inoculation significantly reduces the proline level when comparing it with noninoculated tests. Indeed, in the control, a Proline content equal to 0.24 µg g-1FM is recorded, this value is significantly low compared to the inoculated test (0.97 µg g-1FM).
Metal stress significantly increased amino acid (AA) content in treated samples. Without inoculation, AA levels more than doubled (200.66%) at 2 g L-1Cd(II) compared to the control. Under Cu(II) stress, AA content increased proportionally with concentration, reaching 0.135, 0.185 and 0.19 µM equiv leucine g-1FM at 1.5, 2 and 3 g L-1, respectively. In contrast, in the combined metal treatment, AA content increased as metal concentrations decreased ( Table 3 ).
Inoculation of seeds by Bradyrhizobium sp. ( Lotus ) reduces A.A contents significantly. In fact, we note a significant drop from 8.37 to 18.66% in AA content. for all concentrations compared to non-inoculated tests. We also note that the AA contents increase with the increase in the concentration of the medium in Cd(II) and associated TM.
In non-inoculated seeds, total soluble sugar content increased significantly in all treatments with individual metals and their mixtures compared to the control (3.74 µg g-1FM). Sugar levels rose with increasing metal concentrations. In contrast, inoculation with Bradyrhizobium sp. ( Lotus ) significantly reduced total soluble sugar content compared to non-inoculated seeds ( Table 4 ).
In the absence of bacterial inoculation, very low amylase activity is noted of the control (5.74 x 10-1U ml-1). The α-amylase content increases slightly with the increase in the concentration of Cd(II) and Cu(II) where the increase in the concentration of the mixture of the two elements causes a drop in α-amylase and reaches 2.55 x 10-5U ml-1( Table 5 ).
Inoculation of trefoil seeds with Bradyrhizobium sp. ( Lotus ) significantly increased α-amylase activity compared to non-inoculated controls, which showed 61.1×10-1U ml-1. Amylase activity decreased progressively with increasing Cu(II) concentration, with the lowest activity observed in the Cu(II)+Cd(II) mixture treatments (0.97×10-1U ml-1at concentration III).
Without bacterial pre-inoculation of the trefoil seeds, low DNA contents were noted in the treated tests compared to the control (26.35 µmol g-1DM) except for concentration II of the medium containing the mixture Cu(II)+Cd( II) where the DNA content increases significantly by 58.56% (41.76 µmol g-1DM) ( Table 6 ).
In the absence of Bradyrhizobium sp. ( Lotus ), DNA content was notably low, with slight increases observed at higher TM concentrations. Inoculation with Bradyrhizobium sp. ( Lotus ) significantly enhanced DNA content across all metal treatments compared to the control (29.23 µmol g-1DM). However, DNA levels in trefoil seeds decreased significantly with increasing concentrations of individual metals and their mixtures.
Proline constitutes less than 5% of the total free amino acids, it is recognized as one of the most prevalent stress metabolites ( Matysik et al ., 2002 ; Sharma and Dietz, 2006 ; Lei et al ., 2007 ). It plays multiple roles, including osmoregulation, chelation and detoxification of heavy metals, enzyme protection, cytosolic pH regulation, stabilization of protein synthesis machinery and scavenging of reactive oxygen species (ROS) ( Sharmila et al ., 2002 ).
In our study, proline content increased under cadmium (Cd) exposure, consistent with reports that Cd induces proline synthesis in various plants, including radish cotyledons ( Biteur, 2012 ). Similar proline accumulation has been observed in response to other heavy metals, such as Cd in peanuts ( Anuradha and Rao, 2007 ) and Cu in rice and carrots ( Chen et al ., 2004 ; Szafrariska et al ., 2011 ).
Our results indicate that Cd and Cu exposure significantly inhibited α-amylase activity, although treatment with a specific Cu/Cd mixture concentration stimulated the enzyme. These findings align with Mihoub et al . (2005) , who reported a marked decrease in amylolytic activity in the presence of these metals. The observed inhibition may result from direct interactions of Cd and Cu with α-amylase or its regulatory proteins, impairing carbohydrate mobilization during germination.
Additionally, DNA content in Lotus ornithopodioïdes L. cotyledons decreased under metal stress, indicating increased sensitivity to heavy metals. This observation aligns with previous studies reporting reduced DNA content in plants such as mustard and beans exposed to heavy metals under saline conditions ( Chatterjee et al ., 1985 ; Bouzid, 2009 ). Lower DNA levels suggest impaired cellular development and protein synthesis, which are critical for plant growth and stress tolerance ( Bouzid, 2009 ).
Inoculation with Bradyrhizobium sp. ( Lotus ) led to a significant reduction in total amino acids, proline and soluble sugars in the presence of copper or cadmium ions. This contrasts with previous findings by Silini et al . (2016) , which indicated an increase in osmoprotectants in wheat seeds after inoculation with Azotobacter under abiotic stress.
The presence of cadmium (Cd) and copper (Cu) adversely affected germination and various biochemical parameters; however, the inoculated seeds exhibited improved performance compared to non-inoculated controls. Specifically, the inoculation led to a reduction in proline and amino acid levels, while promoting higher α-amylase activity and DNA content, indicating a more robust metabolic response to metal stress.
These findings suggest that Bradyrhizobium sp. ( Lotus ) plays a critical role in mitigating the negative effects of heavy metals on plant germination and growth. The mechanisms by which this bacterium enhances resilience may involve the modulation of osmoprotectants and enzymatic activities, which are essential for maintaining cellular homeostasis under stress conditions. Overall, this research highlights the potential of using Bradyrhizobium sp. ( Lotus ) as a biotechnological tool in sustainable agriculture to improve the resilience of crops in metal-contaminated environments.
This research was funded by la Direction Générale de la Recherche Scientifique et du Développement Technologique (DGRSDT) and the CRAPast, Algeria.
The authors extend their acknowledgments to la Direction Générale de la Recherche Scientifique et du Développement Technologique (DGRSDT), Algeria.
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