Implementation of Bacteriogenic Manganese Oxide Nanoparticles to Manage Biotic and Abiotic Stresses

Abstract

Post-harvest diseases, metal stresses and fungal infections are significant yieldlimiting factors affecting a variety of crops. Various techniques are being employed to address these challenges. The economy of Pakistan heavily relies on crop and fruit production, making it essential to tackle these issues with modern techniques to enhance plant and fruit yield. In the past decade, nanotechnology has been reported to offer innovations in the food and agricultural sectors, offering effective solutions to plant ailments. By harnessing the potential of nanotechnology, we can significantly advance agricultural practices and ensure sustainable growth in this vital industry. This study has been planned for the biological synthesis and application of manganese oxide nanoparticles (MnO NPs) on tomato to control its fruit rot disease, Pb stress, and fusarium wilt. For better understanding, this study has been presented in three sections. In the first section, surveys were conducted to collect tomato fruit with usual symptoms of brown rot. Diseased fruit parts were excised and cultured on potato dextrose agar (PDA) media plates. After 3-5 days, mycelial mass could be observed in Petri plates and based on its morphological, microscopic and molecular analyses, it was identified as Aspergillus nigar. To control the spread of this disease, manganese oxide nanoparticles (MnO NPs) were prepared in bacterial broth-culture. Bacillus subtilis was grown at 45°C, to induce mild stress of high temperature and its filtrate was obtained and mixed with the solution of manganese acetate. Calcination was performed for the synthesis of MnO NPs, which were later characterized. Fourier transform infrared (FTIR) spectrum revealed the presence of few important stabilizing and reducing agents (carboxylic acid, alkenes, and alkyl halides) on the surface of MnO NPs. X-Ray diffraction (XRD) analysis revealed the size (39 nm) and crystal-like nature of synthesized MnO NPs. Energy-dispersive Xray spectroscopy (EDX) described the mass percentage of manganese (26.4%) and oxygen (23.3%). Scanning electron microscopy (SEM) displayed nearly spherical shape of MnO NPs and confirmed their nano-size. Successfully synthesized MnO NPs exhibited significant mycelial growth inhibition of A. niger and notable control of tomato black rot disease. NPs-treated fruit maintained better biochemical composition and greater organoleptic properties than untreated fruit, and sustained higher percentage of soluble solids, total sugars, reducing sugars and fruit firmness. These results not only proved the usefulness of bacteria supplemented MnO NPs but to our knowledge, this is the first study of tomato black rot, caused by A. niger in Pakistan. In the second part of this study, the effects of nanoparticles were further investigated on tomato plants. This segment focused on assessing the impact of lead stress on plant growth and development. Moreover, this study provided valuable insights into how nanoparticles can mitigate the adverse effects of heavy metal toxicity. Lead (Pb) is a well-recognized toxic trace element that is harmful for both plants and humans. In this study, the nano-priming of tomato seeds was carried out by the fabricated nanoparticles (NPs) to mitigate Pb stress in tomato seedlings. Before sowing under Pb stress conditions, tomato seeds were primed with three different concentrations (0.25 mg/ ml, 2.5 mg/ ml and 5.0 mg/ ml) of MnO-NPs. Synthesized MnO-NPs were characterized. Fourier transform infrared spectrum (FTIR) described the presence of carboxylic acid, alkenes, and alkyl halides on the surface of MnO-NPs, that serve as stabilizing and reducing agents. X-ray diffraction revealed the average size (22 nm) and crystalline structures of MnO NPs while scanning electron microscopy (SEM) also confirmed the nano-flower structure of MnO-NPs. The energy dispersive X-Ray (EDX) analysis determined the mass percentage of manganese (38.27%) and oxygen (36.91%). In comparison to control, application of different concentrations of MnO-NPs improved growth attributes of tomato seedlings including shoot and root lengths, plants fresh and dry weights, chlorophyll and carotenoid contents, relative water content, and proline and sugar accumulation. Nano-priming also decreased relative electrolyte leakage and the production of malondialdehyde and hydrogen peroxide. Seed priming positively influenced tomato seedling and enhanced the gene expression of chalcone synthase and phenylalanine ammonia lyase enzymes, while the expression of chlorophyllase was reduced. These findings suggested that MnO-NPs help tomato seedlings to resist the absorption and accumulation of Pb in roots and leaves. In the third and last section of this study, MnO NPs were applied to control the fusarium wilt of tomato. Fusarium oxysporum f. sp. Lycopersici is the most demolishing myco-pathogen of tomato plant. In this study, MnO NPs were again synthesized using Bacillus subtilis and applied to control Fusarium wilt of tomato. Fourier transform infrared (FTIR) spectroscopy of MnO NPs showed the presence of carboxylic acid, alkenes, alcohols, esters, ethers and alkyl halides on their surface, that serve as stabilizing and reducing agents. UV–vis spectroscopy at 420 nm indicated the synthesis of NPs. Xray diffraction (XRD) determined their average size (28 nm) while scanning electron microscopy (SEM) displayed spherical structure of MnO NPs. The energy dispersive Xray (EDX) analysis revelaed the highest presence of manganese and oxygen. For mycelial growth inhibition of F. oxysporum, three different concentrations of MnO NPs (0.5 mg/mL, 2.5 mg/mL, and 5.0 mg/ mL) were tested, in vitro. Among these, 2.5 mg/mL concentration exhibited best results, and it was used further for the priming of tomato seeds in pot experiment (in vivo). Findings of pot experiment revealed significant control of Fusarium wilt of tomato by the application of MnO NPs (88%), in comparison to control. These plants also displayed notable improvement in growth attributes like shoot and root lengths, plant fresh and dry weights, chlorophyll, carotenoid and relative water content, and proline and sugar accumulation. Nano-priming also improved antioxidant enzymatic activities of superoxide dismutase (SOD) and peroxidase (POD) and decreased relative electrolyte leakage and the production of malondialdehyde and hydrogen peroxide. A histopathological assay revealed severe damage of root and shoot tissues by the application of F. oxysporum. Contrarily, the application of MnO NPs rescued the plant vascular system. These findings suggest a strong role of MnO NPs in controlling the most devastating wilting disease of tomato. Conclusive findings of this study elaborated the successful deployment of Bacillus-MnO nanocomposites for controlling different biotic and abiotic stress conditions. These MnO NPs proved very effective in mitigating fruit rot, Fusarium wilt and Pb stress. These outcomes mark a significant step toward sustainable agriculture, reducing dependence on chemical fungicides and minimizing environmental risks.