An In Vitro Study of the Nature of Protective Activities of Copper Sulphate, Copper Hydroxide and Copper Oxide Against Conidia of Venturia inaequalis

Copper is one of the oldest fungicidal agents, yet little is known about its mode of action. Solubilities of copper fungicides currently in use range from nil (copper oxide) to high (copper sulphate). We have investigated the effect of water solubility on protective activities of copper sulphate, copper hydroxide (slightly soluble) and copper oxide using conidia of Venturia inaequalis and an in vitro test system based on isolated apple leaf cuticles. Effects were assessed microscopically using two vital stains; fluorescein diacetate (FDA) indicates esterase activity and integrity of the spore membrane, and cyanoditolyl tetrazolium chloride (CTC) was included to test the integrity of the mitochondrial respiration chain. Solutions (copper sulphate) or suspensions (copper oxide, copper hydroxide) at concentrations of 1, 10 or 25 mmol/l was applied to the surfaces of cuticles 24 h before inoculation and allowed to dry. Conidia applied to these residues failed to germinate at all treatment concentrations. With copper hydroxide and copper oxide some ungerminated conidia exhibited some residual fluorescence when stained with FDA, especially at low concentrations. At all concentrations tested, ungerminated spores did not fluoresce when stained with CTC. This is the first evidence that copper fungicides kill scab spores by inhibiting mitochondrial respiration. A filtrate prepared from copper oxide suspension was completely ineffective, while a filtrate from copper hydroxide suspension slightly inhibited spore germination and prevented the formation of appressoria. The results indicate that the fungicidal action of the slightly and insoluble copper hydroxide and copper oxide, respectively, cannot be explained based on the concentration of dissolved copper ions. It is likely that solubilization of copper from solid residues was assisted by direct contact between spore membranes, exudates and the solid copper containing particles. The generation of reactive oxygen species (ROS) in suspensions of copper hydroxide and copper oxide could not be detected using chemiluminescence analysis. Hence ROS were not formed on the surfaces of copper containing particles and could not be involved in copper toxicity.