## Results and Discussion
### Characteristics of the Plasma-Activated Water Produced by the Soft Plasma Jet
According to the proteomic study by [PERSON] et al., cellular membrane damage was not the main reason for \(H_{2}O_{2}\)-induced death of _Penicillium expansum_, a food spoilage fungus [58]. Their results suggest that mitochondrial impairment due to the functional alteration of  
\[2H_{2}O+M^{+}\to H^{+}(H_{2}O)+OH+M\,, \tag{3}\]0 Typical SEM images of (**a**) PAW-untreated and (**b**) PAW-treated _A. brasiliensis_ spores. The spores were treated by the PAW for 30 min. Arrows in the SEM image of (**b**) indicate the holes formed on the surface of the fungal spore cell walls. oxidative stress-sensitive proteins is associated with fungal death caused by \(H_{2}O_{2}\). Since we detected \(H_{2}O_{2}\) and \(NO_{x}\) in PAW-3 and PAW-6, we deduced that \(H_{2}O_{2}\) and \(NO_{x}\) in the PAW passed through the opened pores in the cell wall to enter the spore cells and to react with oxidative stress-sensitive intracellular components (DNA, proteins in mitochondria) and eventually bring about spore cell death. This deduction would describe one of the mechanistic processes that lead to the inactivation of fungal spores. Electrophoretic Analysis of Genomic DNA Extracted from the A. brasiliensis Spores Treated with Plasma-Activated Water  
Genomic DNA is chromosomal DNA that governs the function of cells and growth. In fungi, it is enclosed in a nucleolus with a protective membrane. Section 3.3 demonstrates that RONS can reach intracellular components such as protein, nucleic acids, etc. Thus, it is expected that RONS could react with genomic DNA and damage it. To examine whether PAW treatment leads to the degradation of genomic DNA, we extracted genomic DNA from _A. brasiliensis_ spores with and without PAW treatment (negative control). The PAW-1, PAW-2, and PAW-6 were used for the fungal spore treatment. We also examined genomic DNA from the _A. brasiliensis_ spores with direct plasma treatment for 3 min as positive control for DNA damage. The extracted DNAs were analyzed by agarose gel electrophoresis (Figure 8). The genomic DNA of the negative control showed a high molecular DNA band, and some of the DNA bands ranged from 1.5 kb to around 0.25 kb, which are normal in fungal genomic DNA preparation [19]. The genomic DNA of PAW-1 treated spores showed a similar band pattern to that  
\[2H_{2}O+M^{+}\to H^{+}(H_{2}O)+OH+M\,, \tag{3}\]1 **(a)** Flow cytometry measurements of fluorescence intensity for the PAW-untreated and PAW-treated _A. brasiliensis_ spores. **(b)** Gated fluorescence intensity for the PAW-untreated and PAW-treated spores. The black, blue, red, and green-colored curves represent the fluorescence intensities of the control, PAW (1 min), PAW (3 min), and PAW (6 min)-treated spores, respectively. The PAW-untreated and PAW-treated spores were stained with a membrane-impermeable fluorophore, propidium iodide (PI), to investigate the effect of PAW on cell wall integrity. of the negative control. However, the genomic DNA from the spores treated with the PAW-3 and PAW-6 did not show the high molecular DNA band and only showed smeared DNA bands of around 0.25 kb. This result agreed with the electrophoresis pattern of the genomic DNA of the positive control. This agarose gel analysis demonstrates that the PAW treatment with a plasma activation time of 3 min or more could damage the genomic DNA of the _A. brasiliensis_ spores, and the electrophoresis pattern of the damaged genomic DNA by the PAW is similar to that by plasma jet. The genomic DNA damage of spores by the PAW-3 treatment could contribute to sharp reduction of spore viability (Figures 4a and 7). Overall, these results indicate that PAW possesses the same fungicidal ability as the plasma jet. This is the first report to demonstrate that PAW can damage the genomic DNA of fungal spores. It is reported that the oxidative damage by RONS involves DNA strand breaks and modifications of nucleobases, deoxyribose, and nucleotides [59; 60; 61; 62]. Thus, we believe that the RONS contained in the PAW work similarly against the spore DNA of _A. brasiliensis_.