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Adithan Aravinthan,1,* Muthusamy Govarthanan,2,3,* Kandasamy Selvam,4 Loganathan Praburaman,2,3 Thangasamy Selvankumar,3 Rangachari Balamurugan,1 Seralathan Kamala-Kannan,2 Jong-Hoon Kim1 1College of Veterinary Medicine, Biosafety Research Institute, Chonbuk National University, Jeonju, South Korea; 2Division of Biotechnology, Advanced Institute of Environment and Bioscience, College of Environmental and Bioresource Sciences, Chonbuk National University, Iksan, South Korea; 3PG and Research Department of Biotechnology, Mahendra Arts and Science College, Kalippatti, Namakkal, Tamil Nadu, India; 4Centre for Biotechnology, Muthayammal College of Arts and Science, Rasipuram, Namakkal, Tamil Nadu, India *These authors contributed equally to this work Abstract: A rapid, green phytosynthesis of silver nanoparticles (AgNPs) using the aqueous extract of Helianthus tuberosus (sunroot tuber) was reported in this study. The morphology of the AgNPs was determined by transmission electron microscopy (TEM). Scanning electron microscopy–energy-dispersive spectroscopy (SEM–EDS) and X-ray powder diffraction (XRD) analysis confirmed the presence of AgNPs. Fourier transform infrared spectroscopy (FTIR) analysis revealed that biomolecules in the tuber extract were involved in the reduction and capping of AgNPs. The energy-dispersive spectroscopy (EDS) analysis of the AgNPs, using an energy range of 2–4 keV, confirmed the presence of elemental silver without any contamination. Further, the synthesized AgNPs were evaluated against phytopathogens such as Ralstonia solanacearum and Xanthomonas axonopodis. The AgNPs (1–4 mM) extensively reduced the growth rate of the phytopathogens. In addition, the cytotoxic effect of the synthesized AgNPs was analyzed using rat splenocytes. The cell viability was decreased according to the increasing concentration of AgNPs and 67% of cell death was observed at 100 µg/mL. Keywords: cytotoxicity, Helianthus tuberosus, nanobiotechnology, phytosynthesis, splenocytes