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IntroductionPenicillium Digitatum (PD) and Penicillium italicum diseases pose significant economic challenges to citrus fruit production across the globe. The primary aim of this research is to investigate the synergistic effects of low concentrations of H2O2 solution combined with transient spark discharge plasma on the inactivation of PD. Additionally, assess the chemical and physical properties. Ultimately, this approach can be presented as an eco-friendly solution for rinsing citrus fruits on an industrial scale.Materials and MethodsThe Penicillium digitatum (PD) isolate (ATCC 24692) was obtained from the Tehran Molecular Mycology Laboratory and cultured on Sabouraud Dextrose Agar medium at pH 5.6 and 27°C for 7 days. The initial concentration of spores in the solution was determined using a UV absorption spectrophotometer, set to 0.1 at a wavelength of 420 nm, and the concentration of spores was approximately equivalent to 106 spores per milliliter (Palou et al., 2002). In this study, the plasma reactor had a point-to-plane geometry. The high-voltage needle electrode was placed above a Petri dish filled with a microbial solution combined with H2O2, while the grounded electrode was immersed in the solution. The distance between the tip of the needle electrode and the surface of the solution was 15 mm. Solutions of 0.05%, 0.1% and 0.5%v/v H2O2 (35% soluble in water) were added to the microbial solution before plasma treatment. The final volume of the solution was 5 ml and exposure times were 2.5, 5, 10, and 15 minutes. The reactor was fed with an air flow of 2 l/min. A transient spark discharge was generated, characterized by a discharge voltage of approximately 18 kV, short durations of less than 100 ns, and high current pulses exceeding 1A, with a repetition frequency ranging from 0.5 to 10 kHz. After treatment, H2O2, NO2‾, and NO3‾ as the main long-lived species in plasma-activated solution are measured. Also, physical factors such as electrical conductivity and pH were measured. Data Analysis performed using SAS 9.4 software. Results and DiscussionWith increasing plasma treatment time and H2O2 concentration, the log reduction increased across all treatments. The combination of 0.1 and 0.5% H2O2 solution with plasma resulted in complete inactivation of P. digitatum within just 15 minutes. In plasma-treated solutions, regarding chemical properties, the concentrations of H2O2, NO2‾, and NO3‾ increased linearly with the treatment time. Furthermore, the electrical conductivity increased linearly, with a notable acceleration in the treated 0.5% H2O2 solution, reaching 373µS cm-1. Additionally, pH value dropped from an initial value of 6.95, using distilled water as a control, to a low of 2.14 for plasma treated with 0.5% H2O2 after 15 min of exposure.ConclusionThe combined treatment was more effective than the isolated use of hydrogen peroxide solution. H2O2 enhances the effectiveness of plsma sterilization without requiring additional power input. Consequently, the synergistic application of atmospheric pressure plasma and H2O2 proved to be a promising method for the inactivation of PD. The findings indicate that reactive oxygen species (ROS) significantly contribute to the inactivation of PD cells, as well as the concentration of H2O2. Finally, the combination of H2O2 solution at 0.1 and 0.5% with cold plasma presents an environmentally friendly method for sanitizing citrus fruits.Acknowledgment This work was supported by the Ferdowsi University of Mashhad [Grant number 48527]. The authors greatly appreciate the technical support from Mazandaran University in Iran, particularly from the Department of Atomic and Molecular Physics.