Inactivation effects of plasma-activated water on Fusarium graminearum

The continuous usage of fungicides poses a potential threat to the environment, ranging from mere irritation to being very toxic to human beings and organisms. Plasma-activated water (PAW) has recently gained much interest as a promising candidate to inactivate fungi. However, the inactivation mechanisms of PAW are still not well understood. In this study, the effect of PAW on the viability and the cellular responses of Fusarium graminearum in PAW inactivation were investigated. The results showed that microbial activity of spores was significantly inhibited by PAW treatment (P < 0.05). The symptoms caused by F. graminearum were significantly reduced on the spikelets. Our data indicated that PAW could induce cell wall sculpturing, membrane permeability changes, and mitochondrial dysfunction. Differential gene expression analysis also confirmed that the cell membrane, the cell wall and the mitochondria were the organelles most affected by PAW. The results from this study facilitate the understanding of the mechanisms underlying the responses of F. graminearum to PAW and the development of PAW as a potential fungicidal agent or an effective supplement to fungicides. Highlights The viability of F. graminearum is notably inhibited by PAW The symptoms caused by F. graminearum were significantly reduced on the spikelets Oxidative stress induce cell wall sculpturing, membrane permeability change PAW can cause the mitochondrial dysfunction Cell wall, membrane and mitochondria are the most affected organelles by PAW

For the mycelium growth assay, 2 mL of PAW was transferred into the test tubes containing 0.5 mL 137 of F. graminearum spore suspension. After exposure for 1 h, 10 μl of F. graminearum spore suspension 138 was dropped at the center of CM or CR (Congo red was added to complete medium to 100 µg mL -1 or 139 200 µg mL -1 ) and CFW (calcofluor white was added to complete medium to 50 µg mL -1 ) plates. The agar 140 plates were incubated at 28 °C. Sterile water (2 mL) was used as the control. The colonial diameters of 141 the treated strain were carefully measured by a caliper, every 12 h over 5 d, and the inhibition ratios were 142 calculated using the following formulas: (1) 144 where D c and D t are the colonial diameters of the control and treated groups, respectively.

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The dry weight of the mycelial biomass was measured after 7 days of cultivation in liquid CR or 146 CFW medium. 156 Although germ tube emergence marks the culmination of the germination process, it was used as a 157 convenient marker for germination in our experiments (Semighini et al., 2008). The conidial germination 158 ratios were calculated using the following formulas: where R is the conidial germination ratio, N g is the conidial germination number, and N t is the conidial 161 number.

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The relative conidial germination ratios were calculated using the following formula: where R e is the relative conidial germination ratio, and R t and R c are the conidial germination ratios 165 of the treated and control groups, respectively.  corresponds to larger amounts of hydrogen nitrate and nitrous acid, which lead to a lower pH value.

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The electrical conductivity can indicate the concentrations of free ions present in an electrolytic 248 solution. The electrical conductivity of PAW was measured before and after plasma activation (Fig. 1d).

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The conductivity of water after plasma activation increased from 2.3±0.3 S cm -1 to 430.2±1.1 S cm -1 .

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The electrical conductivity of PAW increased in a plasma activation time-dependent manner, which

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The results showed that the radial growth of F. graminearum was severely (P < 0.05) inhibited after 278 PAW treatment (Fig. 3a). After 12 h of incubation, the mycelium length of PAW15-treated F.  and TEM results showed that spore cell wall and membrane sculpturing occurred after PAW treatment 327 (Fig. 4), supporting this hypothesis.

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In addition to reactive species, the low pH can also inhibit hyphal growth (Wiebe et al., 1996). The indicated a defect in cell membrane integrity (Fig. 4c).

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The leakage of intracellular DNA/RNA and proteins was also a significant indicator of the disruption 354 of cell membrane integrity. As shown in Table 1, upon the addition of PAW to F. graminearum spores,   Mapping of the raw RNA-seq expression data revealed that a total of 2837 (22%) of the 12,792 F.

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graminearum unigenes were differentially regulated by PAW treatment. The thresholds for differential 388 expression were P adjusted < 0.001 and expression fold Change > 5 for up-and downregulated genes.

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By these criteria, we identified 703 genes upregulated and 676 genes downregulated by PAW. As shown 390 in Fig. 6, of the 1379 genes, 944 genes were classified as having "unknown function".

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The most upregulated gene involved in mitochondrial function is listed in

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In line with our main objective of identifying cell wall-, membrane integrity-or mitochondria-431 related genes, thirteen PH-1 unigenes were selected for further analysis on the basis of their expression 432 levels and possible roles in defense mechanisms in response to PAW ( Table 2). As shown in Table 5

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In conclusion, the results from our study confirmed that PAW treatment is a highly effective 441 disinfection procedure against F. graminearum. It has the potential to control fungal contamination. This 442 study also unravels the potential antifungal mechanism of PAW from the perspective of cellular response

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Declaration of Competing Interest:

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The authors declare no conflict of interest in this paper.

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The authors gratefully acknowledge the financial support provided by the National Natural Science