Copper nanoparticles combined with fungicides: an eco-compatible anti-resistance tool against Alternaria alternata

Copper nanoparticles (Cu-NPs) were evaluated as a potential control agent against both sensitive and fludioxonil-resistant isolates of Alternaria alternata in vitro and in vivo. Five highly fludioxonil-resistant, spontaneous mutants of A. alternata, were acquired from wild-type strains after selection on media containing fludioxonil. Mutations in the coding region of the AaHK1 gene leading to premature termination of the protein in resistant isolates were identified by sequencing. Notably, these resistance mutations did not adversely affect mycelial growth or virulence; however, fludioxonil-resistant isolates demonstrated increased sensitivity to osmotic stress and reduced conidia production compared to wild-type strains. Cu-NPs exhibited a superior fungitoxic effect against both wild-type and resistant isolates, outperforming the reference Cu(OH)₂-containing fungicide. The combination of Cu-NPs with fludioxonil or iprodione resulted in a significant synergistic effect, that could be associated with an enhanced fungicide bioavailability. The fungitoxic mechanism of Cu-NPs was not solely attributable to copper ion release, as evidenced by the synergistic interaction with EDTA, a strong chelating agent, and the distinct lack of correlation with Cu(OH)₂. The synergistic activity observed between Cu-NPs and EDTA may be attributed to a reduction in nanoparticle size due to the chelating agent's capping effect. Additionally, ATP-dependent ion efflux may play a role in the fungitoxicity of Cu-NPs against A. alternata, supported by the additive effects observed with fluazinam, an oxidative phosphorylation uncoupler. Collectively, these findings indicate that Cu-NPs represent a viable, alternative fungicide against A. alternata and offer a promising strategy for mitigating resistance when used in combination with fludioxonil or iprodione, ultimately reducing environmental impacts associated with synthetic fungicides.

This study was co-financed by the European Regional Development Fund of the European Union and Greek national funds through the Rural Development Program (RDP/ΠΑΑ) 2014 – 2020, under the call "Cooperation for environmental projects, environmental practices and actions for climate change" (project code: Μ16SΥΝ2-00354).