Abstract
Plasma-activated water (PAW) has emerged as a platform for sterilizing fungal pathogens. In this study, we investigated the influence of PAW on black melanized spores of Aspergillus brasiliensis to explore the mechanism of fungal spore inactivation. PAW was prepared by activating deionized water with a nonthermal atmospheric pressure air plasma jet (soft plasma jet). The concentrations of H2O2 and NOx in the PAW treated by the soft plasma jet for 3 min were 50 μM and 1.8 mM, respectively, and the pH of the PAW was 3.10. The reactive oxygen and nitrogen species (RONS) in the PAW increased with longer plasma activation time. After being treated for 30 min in the PAW with a plasma activation time of 3 min, the spore viability dramatically dropped to 15%. The viabilities of 0.3% H2O2- and 0.3% HNO3-treated spores were 22% and 42%, respectively. The breakage of the spore cell wall by the PAW was revealed in scanning electron microscope images and flow cytometry measurements. Disruption of cell wall integrity provides a path for intracellular components to escape and RONS of the PAW can attack intracellular components directly. Degradation of high molecular genomic DNA was also observed by agarose gel electrophoresis. These results suggest that long-lived reactive species generated in the PAW play an important role in the inactivation of melanized fungal spores. Consequently, PAW produced by a soft plasma jet can be applied to sterilize bioprotective walled fungal spores in a relatively large volume.
Highlights
Fungi are eukaryotic organisms that grow with hyphal cells and produce spores
When the soft plasma jet entered the aqueous solution, additional reactive oxygen and nitrogen species (RONS) could be induced from the interaction between plasma radicals and water molecules, presented in the following reactions [30,31,32,33,34,35,36,37,38]: N2 + e → N + N + e, (1)
When the spores were treated directly with the soft plasma jet for 3 min. This result indicates that the viability of the Plasma-activated water (PAW)-treated spores is almost comparable to that of the spores treated directly with plasma. These results demonstrated that the PAW produced by the soft plasma jet can be applied in fungal spore inactivation
Summary
Fungi are eukaryotic organisms that grow with hyphal cells and produce spores They have evolved diverse morphological and genetic groups that are estimated to include 1.5–5.0 million species on earth [1]. They play key roles in natural ecosystems as material decomposers, mutualists with other organisms, and pathogens of animals, plants, and humans [2]. This study was performed to clarify the influence of PAW on the viability and cell wall integrity of melanized fungal spores For this purpose, we selected Aspergillus brasiliensis (formerly A. niger), a fungal species that produces tremendous amounts of melanin pigmented spores within short time periods and is present in the air and our living environment [24]. To achieve the aim of this study, we first investigated the properties of PAW generated by soft plasma jet, examined the effect of PAW treatment on the viability of A. brasiliensis spores, and compared the effect on spore viability with the effect of chemically-induced RONS (H2 O2 and HNO3 )
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