Fungal diseases are a persistent problem in the cultivation of white button mushrooms (Agaricus bisporus). The chemical control of pathogens is becoming less effective and less desirable, so new ways to limit these infections are urgently required. What is more, the disease is mostly controlled through cultural practices and good hygiene on mushroom farms. The aim of this study was to evaluate the fungicidal effects of ozone on fungal pathogens of common mushrooms. Experiments with the use of ozone gas for disinfection of growing rooms after the completion of the mushroom growing cycle were carried out. The fungicidal effectiveness of ozone fumigation was evaluated on the basis of the survival rate of the spores of the pathogens tested (Lecanicillium fungicola, Cladobotryum dendroides, Mycogone perniciosa, and Trichoderma aggressivum). Spore suspension was applied to aluminum plates and then was exposed to gaseous ozone. The assessment of the growth of colonies of fungal isolates obtained from infected surfaces was carried out using Rodac contact test plates. The results showed that L. fungicola, M. perniciosa, and C. dendroides isolates were sensitive to the gas ozone. In order to achieve 100% efficacy against Mycogone strains, a minimum of 6 hours of ozonation had to be applied, whereas for Cladobotryum strains, a minimum of 8 hours had to be applied. The Lecanicillium species was the most sensitive to ozonation because 30 minutes of ozonation was enough to gain 100% inhibition of its growth. No satisfactory results were obtained in the case of the pathogenic species of Trichoderma, regardless of the experimental conditions. Nevertheless, this study has demonstrated the usefulness of ozone as a disinfectant for empty growing rooms after the completion of the mushrooms’ cultivation cycle.
