Fig 1.
(color online) Schematic view of the devices and treatment setting.
A. Set-up for ozone treatment. Ozone generator was connected to an acryl container filled with fungal spore suspension or rice seeds. B. Set-up for treatment with arc discharge plasma. A high voltage DC power supply was connected to two electrodes submerged in spore or rice seeds suspension in the container. C. Voltage and current profile during arc discharge at 10 kV and 12 Hz.
Fig 2.
Germination of fungal spores after ozone or plasma treatment.
Germination of F. fujikuroi spores after treatment with ozone (A) or arc discharge plasma (B). Germinated fungal spores were appeared as colonies on PDA plate and the number of colonies (CFU) was counted and converted to the log scale. Each value was an average of 3 experimental replicates and indicated on the bar. Student t-test was performed between control and each treatment; ** p < 0.01.
Fig 3.
Structure of fungal spores after ozone or plasma treatment.
Morphology and internal structure of spores analyzed by using SEM (A) and TEM (B) after ozone and arc discharge plasma treatment. Fungal spores in water (107 / ml, total 200 ml) were treated with ozone for 30 sec or arc plasma for 10 min as described in materials and methods. After treatment, spores were collected by centrifugation and processed for electron microscopy.
Fig 4.
(color online) Disinfection of rice seeds by ozone treatment.
A. Percentage of infected seeds after treated by ozone in water for 1 and 2 hrs (upper graph) or in water of different pH (3 and 10) and with different ozone injection rate (1 and 3 lpm) for 2 hrs (bottom graph). In upper graph, each value was an average of 3 experimental replicates. Student t-test was performed between control and each treatment; ** p < 0.01. B. Fungal growth from rice seeds after treated with ozone in water for 1 and 2 hrs. C. Fungal growth from rice seeds after treated with ozone in water of different pH (3 and 10) and ozone injection rate (1 and 3 lpm) for 2 hrs. All plates were incubated at 25°C for 6 days and then imaged.
Fig 5.
(color online) Disinfection of rice seeds by arc discharge plasma treatment.
A. Percentage of infected seeds after treated in water by arc plasma discharged at 10 kV and different frequencies (6, 9, 12 Hz) for 30 min (upper graph) or discharged at 10 kV and 12 Hz for 5, 10, 20, and 30 min (bottom graph). Each value was an average of 3 experimental replicates. Student t-test was performed between control and each treatment; * p< 0.05, ** p < 0.01. B. Fungal growth from rice seeds after treated with arc plasma discharged at 10 kV and different frequencies (6, 9, 12 Hz) for 30 min. C. Fungal growth from rice seeds after treated with arc plasma discharged at 10 kV and 12 Hz for 5, 10, 20, and 30 min. All plates were incubated at 25°C for 6 days and then imaged.
Fig 6.
Measurement of temperature and pH in water after ozone or arc discharge plasma treatment.
A. Temperature of water after treatment with arc discharge plasma. Arc plasma was discharged at 10 kV and 12 Hz for 0.5, 1, 5, and 10 min or at 10 kV and different frequencies (6, 9, 12 Hz) for 30 min. B. pH of water after treated with ozone or arc discharge plasma for indicated time. Ozone injection rate was 1 lpm. Arc plasma was discharged at 10 kV and 12 Hz for 0.5, 1, 5, and 10 min or at 10 kV and different frequencies (6, 9, 12 Hz) for 30 min.
Fig 7.
Level of reactive species in water after ozone and arc discharge plasma.
A. Concentration of ozone (ppm) present in water after ozone injection (1 lpm) for indicated time. All measurements were performed in 3 replicates. Student t-test was performed between each treatment time; ** p < 0.01. B. Relative level of ROS (OH radical, peroxynitrite, singlet oxygen) and RNS (nitric oxide) in water after ozone or arc plasma treatment compared to control (no treatment). All values on y-axis are the ratio between treatment and control (ratio = ODwavelength of ozone or plasma treatment / ODwavelength of control). All measurements were performed in 3 replicates.
Fig 8.
SEM analysis of seed surface after ozone or arc discharge plasma.
A. Surface of rice seeds after treated with ozone (2 h) or arc discharge plasma (10 kV, 12 Hz for 30 min). Rice seeds with no treatment were used as control. Arrows indicate fungal spores. B. Set up for treatment with both ultrasonic wave and ozone in water. Ultrasonic wave was generated at 350 W power and 40 kHz frequency. C. Percentage of infected seeds after treated in water by both ultrasonic wave (indicated as Sonif.) and ozone (O3) for 2 or 3 hrs. Each value was an average of 3 experimental replicates. Student t-test was performed between control and each treatment; ** p < 0.01.