Dynamics of nitric oxide level in liquids treated with microwave plasma-generated gas and their effects on spinach development

In this study, we generated water and phosphate buffer treated with microwave plasma-generated gas in which the major component was nitric oxide (PGNO), and investigated the efficiency of the treated water and buffer in fertilization and sanitation. Real time NO level monitored by an electrode sensor was linearly increased over PGNO injection time, and removal of O2 from liquid before PGNO injection accelerated NO assimilation into liquids. Residual NO was still present 16 h after PGNO injection was stopped. H2O2, NO2−, and NO3− were also detected in PGNO-treated liquids. Spinach plants applied with 10 and 30 times diluted PGNO-treated water and 0.5 mM phosphate buffer showed slightly higher height and dry weight than control after 5 weeks. Plants grown with 10 and 30 times diluted PGNO-treated water exhibited the increased tolerance to water deficiency. Significant anti-microbial activity within 1 h was observed in un-diluted and in half-diluted PGNO-treated water and 0.5 mM phosphate buffer. Our results suggest that water or phosphate buffer containing NO, H2O2, NO2−, and NO3− can be produced by PGNO treatment, and that PGNO-treated water or buffer can be used as a potential fertilizer enhancing plant vitality with sanitation effect.

limitations in using these sources have also been found in terms of efficiency and economic costs [26][27][28] . Plasma (particularly microwave plasma) generated NO can be another potential source for exogenous NO, which can be produced in large quantity with less cost compared to pure NO gas. A study shows that NO dominant in a plasma jet generated using air and DC voltage is responsible for the inactivation of yeast and bacteria 29 . Plasma-generated NO as a potential fertilizer and enhancer of plant vitality has not been actively studied. In particular, fertilization that makes plants stronger to stresses caused by climate change is becoming more important these days than ever. In this study, we investigated the potentiality of water and buffer treated with microwave plasma-generated gas, in which the major component was NO, in enhancing plant vitality and inactivating microorganisms. Particularly, relationship with the chemistry of water and buffer treated with microwave plasma-generated gas was intensely examined in the study.

Results
Level of NO dissolved in water and phosphate buffer. In our study, microwave torch plasma was produced using the mixture of nitrogen and oxygen gas provided with 10 lpm (liter per minute) and 400 sccm (cc per minute), respectively ( Fig. 1a and b). The level of nitric oxide (NO) in gas generated by microwave plasma torch was increased when more oxygen was provided per unit time (Fig. 1c). About 6,500 ppm of nitric oxide (NO) was detected in the plasma-generated gas produced using 10 lpm nitrogen and 400 sccm oxygen (Fig. 1c). Since the majority of gas generated by microwave plasma torch was nitric oxide (NO), we designated the gas as plasma-generated nitric oxide (PGNO) in further description in the text.
After PGNO was injected into 1 L of deionized water or 0.5 and 50 mM potassium phosphate buffer (pH 6.0), the level of dissolved nitric oxide was measured in real time using electrode sensor ( Fig. 1d and e). Since oxygen dissolved in water and phosphate buffer can react with nitric oxide, resulting in the reduction of nitric oxide level in liquids, it was purged by injecting nitrogen gas into water and buffer, before injecting PGNO. The concentration of oxygen dissolved in water was dramatically reduced after the injection of nitrogen gas (Fig. 2a). About 90% of the dissolved oxygen was purged after 10 min of nitrogen gas injection, and the level of dissolved oxygen was not significantly changed after that (Fig. 2a). Likewise, about 90% of dissolved oxygen in both 0.5 and 50 mM potassium phosphste buffer was removed after injection of nitrogen gas for 60 min (Fig. 2a). The NO microsensor  modified with perfluorinated xerogel-derived gas-permeable membrane (i.e., a membrane selectively permeable towards NO) was characterized by 27.5 nA/μM of sensitivity, 0.  Fig. S1). The level of nitric oxide (NO) dissolved in water and phosphate buffer was increased over the injection time of PGNO (Fig. 2b). In water, nitric oxide level was more rapidly increased when oxygen was purged for 60 min (Fig. 2b). In potassium phosphate buffer, the NO level was increased faster with oxygen purging, than without (Fig. 2b). The NO concentration was increased slightly faster in 0.5 mM than in 50 mM phosphate buffer, but the level became similar after PGNO injection for 50 min (Fig. 2b). Generally, the increase in NO level was slightly faster in water than phosphate buffer under similar level of oxygen purging (Fig. 2b).
The level of NO was continuously monitored after the PGNO injection was ceased. After PGNO was injected for 50 min and then ceased, the NO concentration was exponentially decreased over time, and about 15-30 µM NO was detected in water and phosphate buffer after 16 h (Fig. 2c). There was no significant difference in the rate of NO level decrease between with and without oxygen purging (Fig. 2c). The dynamics of NO decay in PGNO injected water was compared to that in pure NO gas (commercially purchased) injected water, in order to determine NO life in 1 L water. Pure NO gas was injected into 1 L water (no oxygen purging), until the dissolved NO level reached that of PGNO injected (for 50 min) water. After the pure NO gas and PGNO injection was stopped, the NO level was decreased slightly faster in PGNO than pure NO gas injected water in the early stage, and then decreased at similar rate in both liquids after 10 h (Fig. 2d). The NO level reached around 20 µM in both pure NO gas and PGNO injected water after 16 h (Fig. 2d).
The pH of both water and 0.5 mM phosphate buffer was dramatically reduced to around 3 after PGNO injection for 10 min, and then no further change was observed during PGNO injection. About pH 3 was maintained up to 2 h after stopping PGNO injection (Fig. 3). In 50 mM phosphate buffer, the pH was slightly reduced during PGNO injection (Fig. 3). The pH was around 5.5 after PGNO injection for 50 min, and similar pH was kept up to 2 h without PGNO injection (Fig. 3). This might be due to the higher buffering strength of 50 mM phosphate buffer than water and 0.5 mM buffer. Oxygen purging did not change the pattern of pH decrease in both water and phosphate buffers (Fig. 3).
Level of other reactive species and ions in water and phosphate buffer. We observed that the NO level was decreased slightly faster in PGNO than pure NO gas injected water in the early stage (Fig. 2d). This suggests that NO has reacted with other species that may be present in PGNO water. To test this hypothesis, we investigated the presence of other reactive and ion species in PGNO treated water and buffer. Hydrogen peroxide (H 2 O 2 ) was detected in PGNO-treated water and phosphate buffer. The H 2 O 2 microsensor electropolymerized with poly(3-aminobenzoic acid) (PABA) permselective membrane (i.e., a membrane selectively permeable towards H 2 O 2 ) was characterized by 3.15 nA/μM of sensitivity, 0.6 μM of detection limit (S/N = 3), and t 95% < 3 s of response time with negligible response towards interfering species (at 50 μM for each species) such as nitric oxide (NO), nitrite (NO 2 − ), nitrate (NO 3 − ), peroxynitrite (ONOO − ), hydroxyl radical (OH), and superoxide (O 2 − ) ( Supplementary Fig. S2). H 2 O 2 level was gradually increased during PGNO injection, but not as rapidly as that of NO (Fig. 4). About 50-65 µM H 2 O 2 was detected in both water and 0.5 mM phosphate buffer after 50 min PGNO injection (Fig. 4). Oxygen purging did not change the H 2 O 2 level in water (Fig. 4). However, in 0.5 mM phosphate buffer, the H 2 O 2 level increased slightly faster without oxygen purging, than with (Fig. 4). The presence of several ions was analyzed in PGNO-treated water and phosphate buffer using ion chromatography. None of the positive ions analyzed was detected in both PGNO-treated water and phosphate buffer, except potassium (from potassium phosphate buffer) ( Table 1). For negative ions, nitrite (NO 2 − ) and nitrate (NO 3 − ) were detected in PGNO-treated water and phosphate buffer, besides phosphate ion (PO 4 3− ) from potassium phosphate (

Effect of PGNO-treated water and phosphate buffer on plant vitality. PGNO-treated (50 min)
water and 0.5 mM phosphate buffer without and with dilution of 2, 10, and 30 times were applied to spinach seeds, and the germination and development were analyzed. The pH of PGNO-treated water and phosphate buffer was generally lower than that of control (no PGNO treatment), ranging from pH 3 to pH 5.5 (Fig. 5a, Supplementary Table S1). The number of spinach seeds germinated after being soaked in PGNO-treated water or phosphate buffer for 30 min increased over time, and about 85% germination was acquired after 3 days (Fig. 5b, Supplementary Table S1). No significant difference in the speed and percentage of seed germination was observed between control and PGNO-treated solutions, and about 85-90% of seeds were germinated after 7 days in all treatments (Fig. 5b).
Seedling growth was monitored after 35 days (5 weeks). The percentage of survived seedling number after 35 days was significantly greater in the treatment with 1/2 and 30 times diluted phosphate buffer (0.5 mM), than in control (no PGNO-treated buffer) (Fig. 6a, Supplementary Table S1). A slightly higher percentage was observed in the treatment with 10 and 30 times diluted PGNO water and 10 times diluted PGNO buffer (Fig. 6a). The shoot length of plants grown for 35 days was significantly increased after the treatment with 10 and 30 times diluted PGNO water and phosphate buffer (Fig. 6b, Supplementary Table S1). Root length was significantly greater in plants treated with 30 times diluted PGNO water and 10 and 30 times diluted phosphate buffer (Fig. 6b, Supplementary Table S1). Average dry weight per plant was slightly higher in the treatment with 1/2, 10, and 30 times diluted PGNO water and phosphate buffer than control (Fig. 6c buffer (Fig. 6c). Plants treated with no diluted PGNO water and phosphate buffer showed lower survival, height, and dry weight than control ( Fig. 6a-d). Figure 6D shows that plants treated with no diluted PGNO-treated water and phosphate buffer have narrower leaves and are poorly grown, compared to control. Plants treated with 10 and 30 times diluted PGNO water and phosphate buffer seem to have slightly larger and more abundant leaves than control (Fig. 6d).
Plants grown under the treatment with PGNO water and buffer were tested for tolerance to drought stress and induction for PR10 expression. PR10 is a stress related gene, and its expression is induced in response to drought, salt, nitrosative, and oxidative stresses in spinach, providing the plants stress tolerance 30 . The average dry weight per plant treated with 10 and 30 times diluted PGNO water was significantly greater than that of control after 10 days under drought stress (no watering) (Fig. 7a, Supplementary Table S1). Plants treated with 1/2, 10, and 30 times diluted PGNO phosphate buffer exhibited slightly higher dry weight than control after growth under no water condition for 10 days (Fig. 7a). The transcription level of PR10 was increased in plants treated with 10 and 30 times diluted PGNO water compared to control although the difference was not statistically significant ( Fig. 7b, Supplementary Table S1). There was no obvious difference in PR10 transcription between control and PGNO-treated phosphate buffer (Fig. 7a). Anti-microbial activity of PGNO-treated water and phosphate buffer. In order to analyze the potential of PGNO water and phosphate buffer for inactivating microorganisms, we incubated bacteria and fungal spores in PGNO-treated water and phosphate buffer. There was a 5-8 log reduction in CFU number for E. coli incubated in un-and half-diluted PGNO-treated water and 0.5 mM phosphate buffer for 1 h (Fig. 8a). PGNO-treated water and phosphate buffer diluted 10 times inactivated about 50% of E. coli cells (0.2-0.3 log reduction in CFU number) within 1 h (Fig. 8a). In the treatment of S. aureus, 7-8 log reduction in CFU number was observed in the treatment with un-and half-diluted PGNO-treated water, and about 0.6 log reduction (about  50% of bacterial cells) in the treatment with 10 times diluted PGNO water (Fig. 8b). The CFU number of S. aureus was reduced up to about 2 log scale in the treatment with un-and half-diluted PGNO-treated 0.5 mM phosphate buffer for 1 h, and up to 0.1-0.4 log scale (30-60%) in the treatment with 10 and 30 times diluted PGNO phosphate buffer, compared to control (Fig. 8b). The number of germinated F. oxysporum spores was significantly reduced (60-80%) when spores were incubated in un-diluted PGNO-treated water and phosphate buffer for 1 h (Fig. 8c). Spores treated in half diluted PGNO-treated 0.5 mM phosphate buffer were germinated at significantly lower rate (20% reduction) than control (Fig. 8c, Supplementary Table S1). However, there was no dramatic difference in the percentage of germination of spores treated with 10 and 30 times diluted PGNO water and phosphate buffer (Fig. 8c).

Discussion
Our results suggest several implications for the dynamics of NO level in PGNO treated liquid. First, the removal of O 2 in liquid is critical for enhancing NO assimilation into liquid. Our results show that NO concentration in liquid was more dramatically increased when N 2 gas was injected longer (for O 2 removal), regardless of the kind of liquid (water or phosphate buffer). Although the removal of O 2 is efficient in increasing NO assimilation, O 2 removal by N 2 injection may not be practical in the agricultural application because of economic expenses and the effect of injected N 2 . PGNO treated liquid without O 2 removal was applied to plants in our experiments. Since the working NO concentration enhancing plant vitality is important and can be obtained without O 2 removal, it may not be essential in a large scale or practical application. Interestingly, NO was more slowly assimilated into higher than lower concentration of phosphate buffer (higher buffering strength). This might be because potassium phosphate can react with NO. In addition, slightly higher deposition of NO in water than phosphate buffer could be also explained by the reaction of NO with potassium phosphate. Secondly, our results showed that NO decay in PGNO-treated water and buffer (after 50 min PGNO injection) followed exponential change, and NO was not completely perished in 1 L liquid after 16 h without further injection. Decrease in NO level was slightly faster during the early stages in both water and phosphate buffer. This may be a result of the reaction of NO with other species in PGNO liquids. Slightly faster decrease in NO level in PGNO than pure NO gas-treated water during the early stages also indicates that species other than NO may be present, and react with NO in PGNO-treated  Ions and reactive species, such as H 2 O 2 , NO 2 − , and NO 3 − , were also detected in PGNO-treated water and phosphate buffer in our study. The most dominant radicals in the microwave nitrogen plasma are excited nitrogen molecules, which exist in abundance in the metastable state of N 2 (A 3 Σ u + ) 31 . Since our NO generation system may not be perfectly sealed, humid air could continuously be assimilated into the system, providing water molecules. Hydroxyl molecules (OH) are generated from the dissociation of water molecules when they are in contact with excited nitrogen molecules, according to (1). Meanwhile, hydroxyl molecules combine together forming hydrogen peroxide molecules (2). The nitrogen monoxide molecules (NO) come into contact with the hydroxyl molecules, forming nitrous acid, HNO 2 (3). On the other hand, the nitrous acid HNO 2 from NO and OH combination is quenched, due to the reaction (4). The nitrogen dioxide (NO 2 ) thus formed may be eliminated through the reaction (5).  , and acidic pH. Treatment time (1 h) may be not sufficient for complete antimicrobial activity. Longer incubation or treatment may increase the level of microbial inactivation because the effect of incubation time in plasma-treated media on microbial inactivation is occasionally observed in studies 34 . However, long treatment is not likely to be practical in many applications. Microbial inactivation and fertilization by PGNO water and buffer can be separately achieved if we consider to apply those to small size of stock (concentrated) nutrient solutions. For example, microbes in a small stock solution are inactivated by direct PGNO treatment, and then the PGNO treated stock solution is diluted for applying to plants.
A promising effect of PGNO observed in our study is that 10-30 times diluted PGNO water significantly increase spinach tolerance to water deficient stress, and induce the expression of PR gene (encoding a defense protein against pathogen attack). Slight increase in the tolerance to drought stress was also observed in plants treated with 10-30 times diluted PGNO buffer although the difference was not significant. These results may provide useful information on stress regulation, particularly in these days in which global warning becomes a major issue worldwide. The elevated tolerance and PR10 gene expression may be a consequence of the enhanced plant growth and vitality by PGNO solutions. On the other hand, PGNO itself can trigger defense signaling in plant, since NO is a well-known signaling molecule in the regulation of drought and pathogen stresses [22][23][24] . Expression of many stress related genes can be induced by intracellular and exogenous NO 22 . NO causes S-nitrosylation of proteins such as transcription factors and other signaling pathway proteins, and S-nitrosylated (functionally activated) proteins may trigger or repress the expression of stress related genes such as PR, leading to the promotion of plant tolerance [35][36][37] . Intruiguingly, our results show that PGNO treated water seems to induce tolerance to drought stress and PR10 expression more greatly than PGNO treated buffer. Level of NO is not likely to be a reason because no significant difference is observed between PGNO treated water and buffer. Since potassium phosphate is known to play a role in stress regulation in plants 38,39 , additional potassium phosphate may possibly reduce the effect of PGNO as a stress signal. Further study on this will be needed.
In conclusion, our results suggest that PGNO treatment may be able to help sanitation of water and buffer and enhance the fertilizing efficiency of water and buffer, by which plants can be more tolerant to stresses. PGNO (microwave plasma-generated gas) can be efficiently used in agricultural water management, applicable Scientific RepoRts | (2019) 9:1011 | https://doi.org/10.1038/s41598-018-37711-3 to hydroponic culture system and plant factory. For example, microbial contamination in water or even plant nutrient stock solution is removed by PGNO treatment, and then the sanitated water or nutrient stock solution is diluted and used for plant fertilization. However, action mechanisms of PGNO are still not clear. Our results suggest that NO and NO 3 − may have contributed to the enhancement of plant growth and stress tolerance, and H 2 O 2 and NO 2 − may play more role in antimicrobial effect.
Microwave plasma torch system and treatment of water and buffer with plasma generated gas. Figure 1a and b show the microwave plasma system generating nitric oxide, and treatment of liquids with gas generated from plasma system. The system configuration has been well described in previous study 40 . Microwave (2.45 GHz) radiated from a magnetron passes through a circulator, power monitor, and three-stub tuner, and then is guided through a tapered waveguide, entering a discharge tube made of quartz. In order to generate a microwave plasma torch, nitrogen (10 lpm) and oxygen (400 sccm) gases controlled by mass flow meter were mixed and injected into the system, and a microwave power of 400 W was applied. In the discharge tube, a plasma torch (temperature; 6,000 K; plasma density, 10 13 /cm 3 ) was generated. Gas generated from the torch flame was cooled down by passing through metal pipe wrapped with water tubing (Fig. 1a), and then injected into 1 L of deionized (DI) water or 0.5 and 50 mM potassium phosphate buffer (pH 6.0), as shown in Fig. 1a and b. Phosphate buffer of pH 5.0-6.0 is one of common buffers used in many plant nutrient and tissue culture solutions. We used potassium phosphate buffer rather than plant nutrient solution because nutrient solution contained many components, and the chemistry of solution would be more complicated after PGNO treatment. Therefore, we chose to start with a simple background buffer of plant nutrient solution, potassium phosphate buffer (pH 6.0) to make the system simpler.

Measurement of gaseous nitric oxide level, dissolved oxygen (DO) level, and pH.
The concentration of nitric oxide in gas generated by microwave plasma torch was measured using nitric oxide gas measuring instrument (UniGas 1000+, EUROTRON, Italy). The level of dissolved oxygen (DO) in liquids was measured using dissolved oxygen meter (PDO-519, Lutron electronic, Taiwan). The pH of liquids was measured using a portable pH meter (Eutech Instruments, Singapore). All measurements were repeated at least three times. Ion analysis. Several positive and negative ions in liquids treated with plasma-generated gas were quantitatively analyzed using ion chromatography. After plasma generated gas was injected into 1 L of DI water and 0.5 mM potassium phosphate buffer without O 2 purging for 50 min, treated water and phosphate buffer were filtered (0.5 μm pore size). Filtered liquid was then analyzed using chromatograph ICS-3000 (Thermo Scientific Dionex, Sunnyvale, CA, USA Assay for seed germination and growth. Plasma-generated gas (PGNO) was injected into DI water (1 L) and 0.5 mM potassium phosphate buffer (pH 6.0, 1 L) without O 2 removal (N 2 injection) for 50 min. DI water and phosphate buffer treated with plasma-generated gas were then immediately diluted 2, 10, and 30 times. In order to reduce the time consumed for the dilution process, treated water and buffer were immediately added into new DI water or 0.5 mM phosphate buffer already placed in a 100 ml scaled beaker. Non-treated DI water and 0.5 mM phosphate buffer were used as control. Non-treated (control) and treated (1x, 1/2x, 1/10x, 1/30x diluted) water and buffer were applied to seeds and plants.
For germination assay, spinach seeds (50 seeds per treatment) were soaked in 10 ml of non-treated DI water (control), non-treated 0.5 mM phosphate buffer (control), treated (1x, 1/2x, 1/10x, 1/30x diluted) DI water, or treated (1x, 1/2x, 1/10x, 1/30x diluted) phosphate buffer for 30 min. After soaking, seeds were placed on 2 layers of wet filter paper in a petri-dish, and the petri-dish was incubated in a plant growth chamber (25 °C, 50% humidity, 16 h light and 8 h dark). The number of germinated seeds was counted every day for a week. Three replicate measurements per treatment were performed.
To analyze the effects on plant growth, 50 seeds (per treatment) were planted in each pot (100 mm diameter × 50 mm height) containing vermiculite, and the pots were incubated in a plant growth chamber (25 °C, 50% humidity, 16 h light and 8 h dark). Every once a week, 100 ml of non-treated DI water (control), non-treated 0.5 mM phosphate buffer (control), treated (1x, 1/2x, 1/10x, 1/30x diluted) DI water, or treated (1x, 1/2x, 1/10x, 1/30x diluted) phosphate buffer was applied to each pot. Additional DI water (100 ml) was applied to each pot once a week to prevent drying. Plants were harvested after 5 weeks, and the number of survived plants was counted. Length of shoot and root was also measured in individual plant. Then, plants were dried at 60 °C for 3-4 days, and total dry weight of plants collected from each pot was measured. The average dry weight of individual plant was calculated by dividing total dry weight by number of plants. Each treatment was performed in 3 replicate pots.
Assay for tolerance to drought stress. Spinach seeds were planted in pots (100 mm diameter x 50 mm height) containing vermiculite (50 seeds per pot), and the pots were incubated in a plant growth chamber (25 °C, 50% humidity, 16 h light and 8 h dark). Non-treated DI water (control), non-treated 0.5 mM phosphate buffer (control), treated (1x, 1/2x, 1/10x, 1/30x diluted) DI water, or treated (1x, 1/2x, 1/10x, 1/30x diluted) phosphate buffer (100 ml each) were applied to each pot once a week for 4 weeks. Pots were additionally watered (100 ml per pot) once a week to prevent drying. Then, seedlings were then kept under no water condition for 2 weeks. Plants were harvested after total 6 weeks, and dried at 60 °C for 3-4 days. The total dry weight of plants collected from each pot was measured, and the average dry weight of individual plant was calculated by dividing total dry weight by number of plants. Each treatment was performed in 4 replicate pots. QPCR for quantifying the transcription level of PR10 gene. The level of PR10 mRNA was measured in spinach plants grown for 4 weeks under treatment with water or phosphate buffer pre-treated with microwave plasma-generated gas. Plants grown for 4 weeks were ground into powder in liquid nitrogen, and total RNA was extracted using the TaKaRa RNAiso Plus kit (TaKaRa Bio, Tokyo, Japan), as described in the previous study 44 . The RNA concentration was measured using a nanodrop (Biotek, Winooski, VT, USA), and then 1 µg RNA was treated with RNase-free DNase (Promega, Madison, WI, USA) at 37 °C for 1 h, to remove genomic DNA. The same amount of RNA (120 ng) was used to synthesize cDNA using the miScriptII PCR System, following the manufacturer's protocol (Qiagen, Valencia, CA, USA). PCR conditions were as follows: 60 min at 37 °C, and then 5 min at 95 °C. PR10 gene was amplified and quantified at every thermal cycle using iQ SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) and the CFX96 TM real time RT-PCR system (Bio-Rad, Hercules, CA, USA). The sequence of primers for amplifying PR10 cDNA was as follows: TGGCGGTCCATACAAGTACA (forward) and AAACGACATCGCCCTTAGTG (reverse). PR10 mRNA level was normalized by the quantity of Actin (a reference gene) mRNA. The primer sequences for amplifying Actin cDNA were GAGGCACCATTGAACCCTAA (forward) and AGGGCGTAACCCTCGTAGAT (reverse). The relative expression level of PR10 mRNA in plants treated with water or buffer pre-treated with plasma-generated gas was expressed as relative ratio compared to that of control.
Test for antimicrobial activity of liquids treated with plasma-generated gas. The antimicrobial activity of water and buffer treated with plasma-generated gas was assessed using 2 bacterial (Escherichia coli, Staphylococcus aureus) and 1 fungal (Fusarium oxysporum f.sp. lycopersici) species. Two or three bacterial colonies were suspended in 1.8 ml LB liquid, and then 10 μl of the suspension was inoculated into 15 ml LB liquid. After inoculation, culture tubes were incubated for 16 and 20 h for E. coli and S. aureus, respectively, with shaking. Then, bacterial cells were pelleted down by centrifugation (5 min at 3,134 × g), washed with sterile saline once, and resuspended in new saline. Optical density (OD) of bacterial suspension was adjusted to 0.1 and 0.4 for E. coli and S. aureus, respectively, in order to get 10 8 bacterial cells per ml concentration. F. oxysporum spores were prepared as follows: 200 ml Vogel's minimal media inoculated with pieces of fungal hypha was incubated at 28 °C for 3-4 days with shaking, and fungal culture was filtered through 3 layers of miracloth to get spores. After washing with deionized water once, spores were resuspended in DI water, to make the concentration 10 8 spores per ml. After the concentration was adjusted, 1 ml of bacterial cell or fungal spore suspension was placed in a microfuge tube, and centrifuged at 3,134 × g for 5 min. After liquid was discarded, bacterial or fungal cell pellets were resuspended in 1 ml of water, or buffer treated with plasma-generated gas. Tubes were incubated for the indicated time, and then 100 μl of serially diluted suspension was plated on LB agar (for bacteria) and Potato Dextrose Agar (PDA, for fungus) plates. Plates were incubated at 37 and 25 °C for bacteria and fungus, respectively, and CFU and germinated spore number were counted after 1-2 days.
Statistical analysis. All data were indicated as average and standard deviation of 3 or more replicate measurements. Student's t test was performed to determine the significance between data points. Significant differences were established at p < 0.05 or p < 0.01 (*denotes p < 0.05, and ** denotes p < 0.01).