Heavy metal mediated phytotoxic impact on winter wheat: oxidative stress and microbial management of toxicity by Bacillus subtilis BM2

Abstract

Heavy metals are toxic environmental contaminants, which severely affect microbial composition and functions and, concurrently, crop production. Due to these issues, the present study focussed on the selection of metal tolerant microbes endowed with metal detoxification abilities and their role in the management and remediation of metal contaminated soils. The metal tolerant bacterium BM2, identified as Bacillus subtilis by 16SrRNA gene sequencing, survived well under metal pressure and tolerated 1600 and 2000 μg mL^-1 of Ni and Pb, respectively. The inhibitory impact of metals on wheat increased consistently with a progressive increase in metal concentration. Deposition of Ni and Pb within root and leaf and oxidative stress were validated by SEM, EDX and CLSM. The overall growth parameters of wheat grown under metal stress were improved following B. subtilis BM2 colonization. As an example, B. subtilis with 195 mg Pb kg^-1 enhanced the length and dry biomass of shoots by 14% and 23%, respectively, over the control. Also, strain BM2 improved the grain yield significantly by 49% at 870 mg Ni kg^-1 and by 50% at 585 mg Pb kg^-1 compared to uninoculated plants. Moreover, B. subtilis BM2 relieved the metal stress on wheat and caused a significant drop in proline and malondialdehyde content and the activities of antioxidant enzymes, like catalase (CAT), superoxide dismutase (SOD) and glutathione reductase (GR). This study, therefore, provided solutions to the metal toxicity problems faced by winter wheat and clearly suggests that the metal detoxification potential of B. subtilis BM2 could be greatly useful in the management of metal polluted soils.

Fig. 1. Panel I shows cell surface morphology alterations revealed by SEM micrographs of B. subtilis BM2 cells treated with 200 μg mL⁻¹ Ni (A) and 400 μg mL⁻¹ Pb (B) and an untreated control (C). Panel II displays the EDX spectra and the percentage of various elements in Ni treated (D), Pb treated (E) and untreated cells (F) of B. subtilis BM2. Panel III represents TEM micrographs of B. subtilis BM2 treated with 200 μg mL⁻¹ Ni (G), 400 μg mL⁻¹ Pb (H) and an untreated control (I). The darker regions marked with red arrows indicate possible areas of metal distribution and localization within treated cells.

Fig. 2. Cellular death of B. subtilis BM2 cells grown with various concentrations of Ni and Pb and stained with acridine orange and propidium iodide and compared with an untreated control.

Fig. 3. EDX and elemental analysis of leaf (A–C) and root (D–F) tissues of wheat. (A) and (B) represent mapping and EDX spectra of leaf tissue treated with 100 μg mL⁻¹ Ni and 200 μg mL⁻¹ Pb, respectively. (D) and (E) represent mapping and EDX spectra of root tissue treated with 100 μg mL⁻¹ Ni and 200 μg mL⁻¹ Pb, respectively. (C) and (F) represent mapping and EDX spectra of untreated leaf and root tissues of wheat, respectively.

Fig. 4. Oxidative stress and cell death in wheat roots stained with propidium iodide (PI) and acridine orange (AO) and treated with 25–400 μg mL⁻¹ each of Ni and Pb and compared with an untreated control. The image shows the number of dead cells, which increased with an increase in metal concentration, as revealed by red fluorescence of PI when observed under CLSM.

Fig. 5. Toxic impact of three doses (mg kg⁻¹ soil) of Ni and Pb (dose rates mentioned within brackets) on the biological parameters of wheat plants and bioremediation by metal tolerant B. subtilis BM2: germination percentage (A); length of roots and shoots (B); dry matter accumulation in roots and shoots (C); total P content within roots and shoots (D). C1 and C4 represent uninoculated and Bacillus inoculated controls, respectively. Values denoted with different letters are significantly (P ≤ 0.05) different according to Duncan's multiple range test.

Fig. 6. Effect of three doses (mg kg⁻¹ soil) of Ni and Pb (dose rates mentioned within brackets) on various parameters of wheat plants and bioremediation by metal tolerant B. subtilis BM2: number of tillers per plant (A); number of spikes per plant (B); number of grains per spike (C); grain yield and grain protein (D); straw yield (E). C1 and C4 represent uninoculated and Bacillus inoculated controls, respectively. Values denoted with different letters are significantly (P ≤ 0.05) different according to Duncan's multiple range test.

Fig. 7. Lethal impact of three doses (mg kg⁻¹ soil) of Ni and Pb (dose rates mentioned within brackets) on chlorophyll, proline and antioxidant defence response detected in the foliage of wheat plants and bioremediation by metal tolerant B. subtilis BM2: total chlorophyll and proline content (A); MDA and SOD activity (B); GR and CAT activity (C). C1 and C4 represent uninoculated and Bacillus inoculated controls, respectively. Values denoted with different letters are significantly (P ≤ 0.05) different according to Duncan's multiple range test.

Fig. 8. Concentration of Ni (Panel A) and Pb (Panel B) within roots, shoots and grains of wheat plants grown with and without inoculum and detected at harvest. C1 and C4 represent uninoculated and Bacillus inoculated controls, respectively. Values denoted with different letters are significantly (P ≤ 0.05) different according to Duncan's multiple range test.