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The recent application of nano copper (Cu) compounds in the agrosystem has shown potential to improve the physiological performance and agronomical parameters of crops. We grew alfalfa (Medicago sativa) in potting mix amended with bulk, nano, and ionic Cu compounds at 80 and 280 mg Cu/kg; then, we evaluated plant performance at physiological and molecular levels. Plants treated with bulk/nano Cu presented better agronomical responses. The P and S content was reduced in bulk and ionic Cu-exposed plants, compared to controls (p ≤ .05). All Cu forms increased the content of Fe and Zn in roots and Fe in leaves, compared to controls (p ≤ .05). Leaf-superoxide dismutase expression was augmented ~27-fold and rubisco mRNA was unaffected in bulk/nano Cu-treated plants, compared to controls (p ≤ .05). Bulk/nano Cu incremented the relative abundance of microorganisms involved in the …