Spore Viability and Cell Wall Integrity of Cordyceps pruinosa Treated with an Electric Shock-Free, Atmospheric-Pressure Air Plasma Jet

Atmospheric-pressure Ar plasma jets are known to be detrimental to Cordyceps pruinosa spores. However, it is not clear what kinds of reactive species are more effective with regard to fungal cell death. Herein, we study which reactive species plays pivotal roles in the death of fungal spores using an electric shock-free, atmospheric-pressure air plasma jet, simply called soft plasma jet. Plasma treatment significantly reduced the spore viability and damaged fungal DNA. As observed from the circular dichroism spectra, scanning electron microscope images, and flow cytometric measurements, cell wall integrity was decreased by reactive oxygen and nitrogen species (RONS) from the plasma itself and the plasma-activated water. Consequently, degradation of the spore cell wall allows RONS from the plasma to reach the intracellular components. Such plasma-induced intracellular RONS can attack spore DNA and other intracellular components, as confirmed by electrophoresis analysis and phosphorylated histone measurement. In addition, weakening of the spore cell wall allowed for the loss of intracellular components, which can lead to cell death. Plasma radicals were investigated by measuring the optical emission spectrum of the soft plasma jet, and intracellular reactive oxygen species were confirmed by measuring the fluorescence of 2′, 7′-dichlorodihydrofluorescein-diacetate (H2DCF-DA)-stained spores. The soft plasma jet generated considerable amounts of H2O2 and NOx but a very small number of OH radicals as compared to the atmospheric-pressure Arplasma jet; this indicates that plasma-induced long-lived reactive species (H2O2 and NOx) play an important role in the weakening of spore cell walls and cell death.