# Inactivation of the Plant Pathogen _Pythium ultimum_ by Plasma-Processed Air (PPA)
## 4 Discussion  
All plasma process parameters applied for PPA treatment in our study resulted in the complete inactivation of the vegetative stage of _P. ultimum_ on PDA agar blocks, potentially also preventing spore formation. No growth was recorded for mycelia in any of the PPA treatments over a period of 14 days. As no growth was detected in all the PPA treatments, the Holm-Sidak comparison during RM-ANOVA post-hoc analysis led to identical differences of mean, \(t\) and \(p\) statistics of the PPA treatments relative to the control and gas control. Furthermore, the growth in the control and the gas control was also comparable in magnitude, also leading to identical values for the Holm-Sidak comparison. Overall, increasing the gas flow or shortening the incubation time did not affect the inactivation efficiency. In future studies, exposure times below 15 min could be tested to identify the shortest exposure time still reaching complete inactivation. This would ensure quick processing of seeds during the pre-harvest preparation and would likely save energy costs. One possible mode of action for the inactivation of hyphae is the destructive oxidation of the cell wall components and/or lipid peroxidation by the reactive species. This finally leads to loss of the cell integrity followed by morphological degeneration. It is also likely that after damage to the cell membrane, reactive species could have penetrated to the cell interior and affected intracellular enzymes and other cellular macromolecules. In future studies, microscopic analysis of _Pythium_ hyphae after PPA exposure would give evidence on this and might also help to identify the lowest gas flow rate and lowest exposure time of PPA where morphological damage and inactivation are detectable. Reactive nitrogen species play a crucial role in the inactivation of microorganisms by oxidation of the cytoplasmic membrane, protein, and DNA [36; 37]. It has been shown that PPA generated by microwave discharge is composed of highly reactive nitrogen species including nitrogen dioxide, nitrous acid, and nitric acid. [PERSON] et al. [34] published the qualitative gas composition of PPA generated by 16 slm flow of compressed air containing mainly six different molecules and radicals: nitrogen monoxide and nitrogen dioxide (NO\(\bullet\), NO\({}_{2}\bullet\)), carbon dioxide (CO\({}_{2}\)), water (H\({}_{2}\)O), nitric acid (HNO\({}_{3}\)), and nitrous acid (HNO\({}_{2}\)). In general, indirect as well as direct treatment by cold plasma can lead to the inactivation of microorganisms including bacteria, viruses, and fungi [20] by morphological degeneration, including damage of the cell envelope structures. Previously published studies also gave evidence for damage of the cell envelope structures using a direct plasma exposure [38], which is among others related to lipid peroxidation of cell membranes and loss of cell integrity [39], and finally also reported necrotic death after direct plasma treatment [40]. In addition, morphological changes were reported for two agriculturally relevant fungal species, _Ascochyla pinodella_ and _Fusarium calmorum_ after direct DBD air plasma treatment [41]. In that study, increasing plasma exposure time led to higher inactivation and disruption of hyphae. Cracks and burst hyphae were visible in microscopic investigations leading to the hypothesis that oxidative reactions by reactive species resulted in the degradation of cell walls. Two mechanisms are discussed in the literature for cell envelop disruption due to plasma exposition of microorganisms. [PERSON] et al. [42] applied a glow discharge and suggested that the rupture of the bacterial cell wall is caused by alterations of its components due to oxidative effects generated by reactive species. Other authors also reported oxidative reactions of cell wall components [43; 44]. Furthermore, [PERSON] et al.