## Results and Discussion
### Characteristics of the Plasma-Activated Water Produced by the Soft Plasma Jet
brasiliensis_ spores were subjected to cell component analysis using high-performance liquid chromatography (HPLC). We confirmed with HPLC analysis that mannitol and glucose exist in _A. brasiliensis_ spores (Figure 5). There were two other peaks in the HPLC chromatogram that are not matched with known standard materials. The materials of the two unassigned peaks need to be analyzed with further study. When we consider oxygen radical scavenging properties of mannitol and glucose, we could not rule out that these compounds in _A. brasiliensis_ spores are expected to scavenge \(H_{2}O_{2}\) and \(NO_{x}\) in PAW. PAW-1 treatment for 30 min did not show notable effects on spore viability. In this case, mannitol and glucose might function as scavengers of RONS in _A. brasiliensis_ spores. Meanwhile, PAW-3 or PAW-6 treatment for 30 min reduced the spore viability of _A. brasiliensis_. In this case, it could be possible that the radical scavenging capacity of mannitol and glucose is not enough to deter a reduction in spore viability. The viability of 0.3% \(H_{2}O_{2}\)-treated spores was 22%, but the viability of 0.3% \(HNO_{3}\)-treated spores was 42%. Spore viability decreased with an increase of \(H_{2}O_{2}\) and \(NO_{x}\) concentrations, as shown in the Figure 4b. Between these reactive species, the spore inactivation effect was higher with \(H_{2}O_{2}\) treatment [40]. Since both \(H_{2}O_{2}\) and \(NO_{x}\) can induce the modifications of intracellular components such as nucleic acids and proteins [40; 43] that lead to a detrimental effect on cell viability, a reduction of the spore viability by \(H_{2}O_{2}\) and \(HNO_{3}\) treatments suggests that \(H_{2}O_{2}\) and \(NO_{x}\) are key factors deciding fungal spore inactivation. The fungicidal effects of the PAW due to plasma-inducedand \(NO_{x}\) could be supported by the data of \[O_{2}+e_{aq}\to O_{2}^{-}\,, \tag{2}\]4 The concentrations of \(H_{2}O_{2}\) and \(NO_{x}\) increase with longer plasma activation times. That is, PAW-3 and PAW-6 contain sufficient amounts of \(H_{2}O_{2}\) and \(NO_{x}\) to inactivate _A. brasiliensis_ spores. \[O_{2}+e_{aq}\to O_{2}^{-}\,, \tag{2}\]5 (**a**) Optical absorption spectra of the PAW in the wavelength region of 30–420 nm and (**b**) the fit of the experimental absorption spectrum of the PAW to the sum of three components (\(HNO_{2}\), \(NO_{2}^{-}\), and \(NO_{3}^{-}\)). The plasma-untreated water is transparent in the wavelength region of 30–420 nm. DI water was activated by the soft plasma jet for 1, 3, 6 and 10 min, respectively. \[O_{2}+e_{aq}\to O_{2}^{-}\,, \tag{2}\]6 Optical absorption spectra of (**a**) nitrite, (**b**) nitrate, and (**c**) PAW in the wavelength region of 190–250 nm. (**d**) Normalized absorption spectra of nitrite, nitrate, and PAW. In (**c**,**d**), the PAW was prepared by activating DI water for 3 min with the soft plasma jet. \[O_{2}+e_{aq}\to O_{2}^{-}\,, \tag{2}\]7 Viability of the _A. brasiliensis_ spores treated by (**a**) the PAW and direct plasma and (**b**) chemically induced reactive oxygen and nitrogen species (RONS) (\(H_{2}O_{2}\) and \(HNO_{2}\)) solutions. In (**a**), PAW (1 min), PAW (3 min), and PAW (6 min) are water activated by the plasma for 1, 3, and 6 min, respectively. Direct plasma treatment times are 1, 3, and 6 min. The contact time of the _A. brasiliensis_ spores with PAW and chemically induced RONS solutions was 30 min. Morphology and Cell Wall Structure of the A. brasiliensis Spores Treated with Plasma-Activated Water  
Cell walls and cell membranes are pivotal in controlling cell survival and death in fungi. Therefore, the development of antifungal reagents has focused on finding substances that could disrupt cell walls and cell membranes or inhibit their synthesis [56]. In previous works with plasma treatment on insect pathogenic fungal spores, we found that cell morphology of the plasma-treated spores was shrunken, ruptured, and flattened, indicating it had changed considerably [21].