İnvestigation of the effect of SİO2 nanoparticles and endophytic bacteria applications on drought stress tolerance in alfalfa (medicago sativa L.) under in vitro conditions

Drought is one of the main abiotic stress factors limiting agricultural productivity worldwide and poses a serious threat to global food security. Nanotechnology offers innovations in many fields such as health, energy, environment, and agriculture by enabling the control of materials at the atomic and molecular level. Endophytic bacteria are beneficial microorganisms that support plant growth and increase resistance to environmental stresses In this study, 40 endophytic bacteria were isolated from Opuntia ficus-indica, Agave americana cacti, and succulents grown in Turkey, which have high drought tolerance. Four isolates were selected from among these bacteria based on their pathogenicity and PGPB properties, and applications were conducted. Additionally, the bacteria were treated with SiO₂ nanoparticles at a higher dose than the application dose, and those that maintained their viability were selected based on the MTT test results. Species identification was performed using 16S rRNA sequence analysis and the results were recorded in the NCBI GenBank (A2: Bacillus wiedmannii PP473246, AT3: Bacillus proteolyticus PP473247, HT1: Lysinibacillus macroides PP473248, H17: Enterobacter hormaechei subsp. xiangfangensis PP473250). The effects of endophytic bacteria and SiO₂ nanoparticles on the physiological and biochemical responses of Medicago sativa (alfalfa) under drought stress induced by PEG-6000 were investigated. Total protein, root-stem length, antioxidant enzyme activities, reactive oxygen species, and lipid peroxidation levels were determined in stressed plants. The applied isolates increased root-stem dry weight and chlorophyll content; they decreased H₂O₂, superoxide anion, and MDA levels. Additionally, they increased CAT, APX, GR, POX, and SOD activities. SEM analysis was performed to determine the localization of bacteria in plant cells. Furthermore, the presence of SiO₂ nanoparticles in the cells of alfalfa treated with the prominent AT3 and H17 isolates was demonstrated using CLSM. The presence of bacteria was confirmed by re-isolation from inoculated plants and 16S rRNA analysis. The results indicate that the combined application of isolated endophytic bacteria and SiO₂ nanoparticles is an effective strategy for enhancing adaptation responses to drought stress and mitigating its adverse effects in alfalfa plants.