## Results and Discussion
### Characteristics of the Plasma-Activated Water Produced by the Soft Plasma Jet
The effects of PAW treatment on _Aspergillus_ fungal cells were studied first in the spores of _A. flavus_[23]. [PERSON] et al. showed that the PAW treatment of _A. flavus_ spores could reduce the viability and metabolic activity of the fungus. However, their study did not observe the morphology of spore cell walls treated with PAW, and thus whether PAW is involved in the occurrence of mechanical damage remains in question. The SEM images of PAW-treated _Aspergillus_ spores in our study are shown in \[O_{2}+e_{aq}\to O_{2}^{-}\,, \tag{2}\]8 There was a prominent change in the cell morphology of the PAW-treated spores compared with that of control spores. SEM images show that the PAW-treated _A. brasiliensis_ spores have porous walls, which are not observed in control spores. The formation of holes on the surface of the spore cell wall was the major deformation and the size of the holes varied little among spores. Despite cell wall and membrane damage, the inner cellular components remained mostly inside the punctured spores. The PAW-treated spores did not show flattened spores. These properties contrast with the changes in spore cell wall morphology observed in direct plasma treatment. Our SEM data clearly show that the PAW treatment could cause fungal spore damage by the opening of cell walls. This result means that with direct plasma treatment, the RONS in PAW function by attacking the cell walls of the _A. brasiliensis_ spores. The size and number of opened holes are sufficient to allow RONS in the PAW to penetrate the cell wall and membrane barriers, react with intracellular components such as nucleic acids, proteins, and other metabolites, and cause malfunctioning, resulting in spore cell death. \[O_{2}+e_{aq}\to O_{2}^{-}\,, \tag{2}\]9 HPLC chromatograms of the cellular extract of the _A. brasiliensis_ spores. G and M represent glucose and mannitol peaks. To verify and measure the level of structural damage in the cell wall and membranes of _A. brasiliensis_ spores, we stained the PAW-untreated and PAW-treated spores with PI dye. The permeability of PI dye molecules through the damaged membranes in dead cells is relatively higher than that through a normal membrane in living cells. As shown in Figure 7, the gated fluorescence intensity of PI-stained _A. brasiliensis_ spores is more intense in the PAW-treated spores than in the PAW-untreated spores. Subpeaks are clearly shown in the PAW-treated samples at higher fluorescence intensity, which were absent in the control sample. In Figure 7b, we present the P2-gated fluorescence intensity, calculated from the flow cytometry spectra of Figure 7a. It indicates a 3-fold increase in the PAW-treated samples compared to that of the control. Enhanced fluorescence in the PAW-treated spores is attributed to the penetration of the PI dye into the intracellular region of spores through the damaged membrane. The gated fluorescence intensity was similar between PAW-3 and PAW-6 treated spores. This indicates that the PAW with a plasma activation time of 3 min or more has the potential to cause sufficient damage in the fungal spores. Thus, these data support the results shown in Figure 6 and indicate that PAW can disrupt the rigid cell wall structure of melanized _A. brasiliensis_ spores. The _Aspergillus_ cell wall is a complex structure composed of a network of polysaccharides (\(\alpha\)-glucan, \(\beta\)-glucan, chitin, and galactomannan) and cell wall proteins. Thus, we recognize that the _Aspergillus_ cell wall is an essential factor in maintaining the shape and integrity of the fungal cell [57]. Our study revealed that PAW induced the loss of cell wall integrity and its protective function in the spores. Because cell membranes control traffic into and out of cellular components, damaged membranes lose their control function and thus allow intracellular components such as nucleic acids, proteins, and other metabolites possibly to leak out. However, we do not know yet what cell wall components were affected by RONS in the PAW.