Elucidating nano-Cu interactions in grapevine leaves: formulation-dependent foliar affinity, uptake, and leaf persistence over time†

Copper from agrochemicals contaminates agroecosystems partly because of its low affinity to leaves. Copper-based nanoformulations (nano-Cu) have been proposed to limit Cu foliar wash-off and topsoil contamination. However, the fate of nano-Cu at the grapevine leaf interface remains underlooked. Can Cu from nano-Cu be taken up and translocate to other plant tissues? Are nanoforms persistent in/on leaves? This study examined the fate of Cu applied as CuSO₄, CuO-NPs bare, or encapsulated into chitosan-protein capsules (ChiBSACuO-NPs). Cu leaf retention, uptake, translocation, and speciation were analyzed after 7 and 25 days using ICP-MS, μ-XRF, and μ-XANES. Leaf adhesion increased for nano-Cu in comparison to CuSO₄. ChiBSACuO-NPs showed the highest leaf affinity despite their micro-size, likely due to electrostatic affinities in slightly acidic leaf surface microenvironments. Nano-Cu persisted on exposed leaf surfaces for 25 days. It did not lead to leaf lesions, unlike CuSO₄, which induced tissue necrosis and the association of Cu with thiol groups in the leaf vasculature. For all treatments, Cu accumulated at the leaf surface or in the first cell layer (mainly associated with epidermis cells), with limited Cu uptake and low Cu translocation to the petiole. Furthermore, Cu translocation to non-exposed tissues was not detected, and the (limited) nano-Cu that was taken up appeared to undergo rapid reduction upon leaf entry. The decreased Cu toxicity to grapevines of nano-Cu indicates that Cu²⁺ release would have to be triggered in the presence of a pathogen to provide antifungal activity. This study suggests that nano-Cu could allow for the targeting of specific leaf surface tissues, thus providing sustained antifungal efficacy with improved biocompatibility compared to CuSO₄ and offering advantages in safer plant protection strategies.