## 4 Conclusions  
The spore viability and cell wall integrity of _Cordyceps pruinosa_ were investigated to better understand the death mechanism of fungal spores by electric shock-free, atmospheric-pressure air plasma jet (i.e., the soft plasma jet). Plasma treatment significantly reduced the spore viability and damaged fungal DNA. This indicates that the viability of _C. pruinosa_ spores can be controlled using a soft plasma jet that produces plasma radicals in the absence of electric shocks. As observed from the CD spectra, SEM images, and flow cytometric measurements, cell wall integrity was decreased  
Figure 9: Electrophoretic analysis of DNA from _C. pruinosa_ spores treated by a soft plasma jet. Lanes M and 1 represent 1 kb DNA marker and control DNA extracted from _C. pruinosa_ spores without plasma treatment, respectively. Lanes 2, 3, 4, and 5 represent DNA extracted from 1 min, 3 min, 6 min, and 10 min, plasma-treated _C. pruinosa_ spores, respectively. by RONS from both the plasma itself and the plasma-activated water. Consequently, spore cell wall degradation provided a path by which RONS from the plasma could reach the intracellular components. Plasma-induced intracellular ROS are able to attack spore DNA and other intracellular components, as confirmed by electrophoresis analysis and phosphorylated histone measurement. In addition, the weakened spore cell walls resulted in a loss of intracellular components, eventually leading to cell death. Plasma radicals were analyzed using the optical emission spectrum of the soft plasma jet, and intracellular ROS were confirmed by measuring the fluorescence of \(H_{2}\)_DCF-DA_-stained _C. prunosa_ spores. In this research, fungal spore inactivation was achieved by the soft plasma jet using air as the plasma working gas. This atmospheric-pressure air plasma jet (i.e., the soft plasma jet) generated considerable amounts of \(H_{2}O_{2}\) and \(NO_{x}\) but a very small number of \(OH\) radicals as compared to the atmospheric-pressure \(Ar\) plasma jet (i.e., the APPJ); this indicates that plasma-induced long-lived reactive species (\(H_{2}O_{2}\) and \(NO_{x}\)) play an important role in _C. prunosa_ spore inactivation. In addition, an atmospheric-pressure air plasma jet allows for sterilization of fungal spores through the miniaturized plasma device. Thus, as demonstrated in this study, DNA damage and cell wall integrity loss by plasma-induced RONS were further clarified using a soft plasma jet. The application of plasma treatment to fungal spores is versatile. Many fugal species that release large numbers of spores into the air are food and indoor air contaminants. Thus, a plasma apparatus with a sterilization function could be used for sanitary purposes in the food industry, medical manufacturing, and semi-conductors. Since an atmospheric-pressure air plasma jet can make it possible to sterilize fungal spores by miniaturized plasma, meticulous application of plasma-induced RONS may be possible. Regarding _C. prunosa_, stress sources, such as light, could activate the production of spores and biological compounds. The red pigment of the fungus is known to have pharmacological activity of anti-oxidant, anti-inflammatory, and anti-allergic effects [32; 33]. Thus, certain controlled levels of plasma radicals have potential as stress sources that trigger the production of spores and biological compounds. To control the level of plasma-induced RONS for such applications, it is necessary to understand the mechanism by which RONS affect _C. prunosa_ spore cells. Furthermore, to be used as potent biological control agents, information on spore survival under diverse stress conditions is important. Thus, the present work elucidated the effects of plasma-induced RONS as a stress source for _C. prunosa_ spores. In general, fungi utilize nonenzymatic antioxidants, such as reddish pigments, proline, trehalose, and polyols, to protect their cells from attacks by oxidative agents [25]. Currently, the roles of nonenzymatic antioxidants have not been thoroughly studied in _C.