Ozone treatment inactivates common bacteria and fungi associated with selected crop seeds and ornamental bulbs

Highlights • Ozone disinfected seed-borne bacteria and fungi from seeds and ornamental bulbs.• Gaseous ozone showed better performance than ozonated water.• Pre-soaking the seeds in water increased the efficacy of ozone sterilization.• Sterilization success of ozone depended on the type of the seed, pathogen, and treatment conditions.


Introduction
Phytopathogens can attack the reproductive parts of plants during the pre-harvest period in the field, or the products during storage, thereby reducing the quality and quantity of seed harvested (Rennie and Cokerell, 2006;Vega et al., 2019;Gaur et al., 2020;Jagtap et al., 2020). Contamination of seeds or bulbs with some groups of pathogenic microorganisms, such as fungi and bacteria, can limit germination and cause diseases in young seedlings, leading to a significant reduction in plant performance (Gitaitis and Ronald, 2007;Chang et al., 2020). Transmission of seed-borne pathogens during the local and international exchange or trade leads to the introduction of new strains of pathogens that are absent previously, which may subsequently result in the emergence of epidemics (Elmer, 2001;Gergerich et al., 2015;Chalam et al., 2020). Furthermore, some seed-borne fungi such as Fusarium sp. and Alternaria sp. pose a notable threat to human and livestock health by producing mycotoxins in the seeds (Lee et al., 2015;Oldenburg et al., 2017). Hence, seed treatment is regarded by plant pathologists as an extremely important attempt for eradicating or minimizing seed-borne pathogens from seeds or bulbs.
Chemical-based seed treatment methods, such as the application of disinfectants, fungicides, and bactericides, are frequently used to reduce incidence of pathogens in seeds (Errampalli et al., 2006;Scott et al., 2020;Lamichhane et al., 2020;Molin et al., 2021). In addition to that, alternative physical treatments such as plasma containing energized atoms, UV light, and hot water (thermotherapy), which are considered much more eco-friendly, have also been attempted (Scott et al., 2019;Mangwende et al., 2020;Waskow et al., 2021). Ozone (O 3 ) treatment is a fast and economic method to decrease the pathogenic microorganism pressure in drinking and wastewaters, food products such as vegetables, fruits and meat, and food processing equipment (Sopher et al., 2009;Miller et al., 2013;Pandiselvam et al., 2019;Ding et al., 2019;Dong et al., 2018;Khanashyam et al., 2022). O 3 leaves no residue in the products as it can easily be decomposed into non-toxic prodhttps://doi.org/10.1016/j.sjbs.2022.103480 1319-562X/Ó 2022 The Author(s). Published by Elsevier B.V. on behalf of King Saud University. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). ucts, mainly diatomic oxygen, and has a very short lifetime, making it an alternative green treatment to conventional chemicals generally used in seed disinfection and other agricultural practices (Guo and Wang, 2017;Mohan et al., 2005). The United States Food and Drug Administration approved the ozone for sterilization of water as a safe treatment in 1982 (FDA, 1982). Besides, it is allowed to use in fresh and processed food in different countries (Tiwari and Rice, 2012). The agricultural applications of O 3 for the management of diseases during the postharvest storage period, decomposing pesticide residues and mycotoxins into nontoxic products, and improving plant growth have attracted attention lately (García-Martín et al., 2018;Chen et al., 2020;Pandiselvam et al., 2020;Liu et al., 2021;Sujayasree et al., 2022). O 3 can be applied to agricultural products or propagating materials in several ways as gas form or ozonated water, which have advantages and disadvantages depending on the case in process and experimental conditions.
The strong potential of O 3 in eliminating or reducing food-borne bacteria and fungi in seeds has been demonstrated in the last years. Escherichia coli, Listeria monocytogenes and Salmonella spp. were eliminated from alfalfa seeds with ozonated water treatment without any negative effect on the germinability of the seeds (Sharma et al., 2002;Kwack et al., 2014;Adhikari et al., 2019). Trinetta et al. (2011) showed potential applicability of gaseous O 3 to reduce S. enterica and E. coli O157:H7 contamination on pre-inoculated tomato, lettuce, and cantaloupe seeds. Although the seed disinfection potential of O 3 is quite high, there are still few studies on the effectiveness of treatments with O 3 on seeds harboring plant pathogens. Ciccarese et al. (2007) reported that imbibed wheat, barley, and pea seeds exposed to gaseous O 3 showed substantial reduction in the population of Penicillium spp., Aspergillus spp., Alternaria spp., and Rhizoctonia spp. with no significant effect on seed germination. It has been demonstrated that the treatment of sunflower seeds with gaseous O 3 reduced the viability of some fungal microorganisms including Alternaria sp., Fusarium sp., Aspergillus sp., and Penicillium sp. (Rodrigues et al., 2015). O 3 has also been shown to be effective in inactivating Xanthomonas oryzae pv. oryzae (Mohan et al., 2005) and Fusarium fujikuroi spores (Kang et al., 2015) in rice seeds. Treatment with O 3 served as a powerful disinfectant in decreasing the pepper mild mottle virus (Stommel et al., 2021), tomato brown rugose fruit virus (Samarah et al., 2021) and tomato mosaic virus (Paylan et al., 2014) in pepper and tomato seeds, respectively. Sterilization performance of O 3 and its effects on seed germination closely depends on the concentration and duration of treatment, the type and structure of the seed, as well as the target pathogen (Mohan et al., 2005;Pascual et al., 2007). Therefore, it is necessary to determine the required concentration of O 3 and exposure time accurately for each plant species depending on the target pathogen (Pandiselvam et al., 2019). Otherwise, O 3 treatments can result in unsuccessful sterilization and, more importantly, loss of germination. Here we aimed to investigate the effect of different concentrations of gaseous O 3 and ozonated water treatments on the inactivation of seed-borne fungal and bacterial pathogens in the seeds of widely cultivated vegetable (tomato, cucumber) and cereal (wheat) and ornamental (tulip, hyacinth, narcissus) bulbs.

Artificial inoculation of seeds
All seeds used in this study were surface sterilized with 70 % ethanol for 1 min followed by a 1 % sodium hypochlorite for 5 min and then rinsed 5 times with sterile distilled water (SDW). After incubation of 24 h either on King's Medium B (KB) (20 g l À1 protease peptone, 1.5 g l À1 K 2 HPO 4 , 1.5 g l À1 MgSO 4 Á7H 2 O, 10 ml l À1 glycerol, 15 g l À1 agar) or Semiselective Clavibacter medium (SCM) (2.62 g l À1 KH 2 PO 4 Á3H 2 O, 0.5 g l À1 K 2 HPO 4 , 0.25 g l À1 MgSO 4 Á7H 2 O, 1.5 g l À1 boric acid, 10 g l À1 sucrose, 0.1 g l À1 yeast extract, 12 g l À1 agar, 100 mg l À1 nicotinic acid, 30 mg l À1 nalidixic acid, 200 mg l À1 cycloheximide), 10 ml of SDW was added on the plates, and cells were dislodged gently by scraping the colony surfaces with a sterile glass rod. The collected bacterial suspensions were diluted to 10 8 , 10 7 , and 10 7 cfu ml À1 for Cmm, Pst, and Psl with SDW, respectively. To obtain microconidia, mycelial plugs (0.9 cm Ø) were transferred from 10 days old fungal culture grown on potato dextrose agar (PDA) to potato dextrose broth (PDB) and incubated 5 days on a rotary shaker at 25°C and 125 rpm. The conidial suspension was filtered through several layers of cheesecloth, washed many times with SDW and diluted to a concentration of 10 7 microconidia ml À1 . Vacuum infiltration was performed as follows; a round membrane filter with a pore size of 0.22 lm (MF-Millipore Ò Membrane Filter) was placed in a sterile funnel (Sartorius Funnels for Combisart TM ) mounted on a flask connected to a vacuum pump. The disease-free seeds were placed in the funnel with 50 ml of bacterial or fungal spore suspension and subjected to a pressure of À600 mbar for 10 min at room temperature. The inoculated seeds were air dried overnight under sterile conditions (Ribeiro et al., 2016;Xiulan et al., 2010;Ochi et al., 2017). Control seeds were treated with SDW using the same protocol. Surface sterilized ornamental bulbs were inoculated with Pcc by immersing the bulbs in a suspension of bacteria (10 6 cfu ml À1 ) for 24 h. Inoculation of wheat seeds with teliospore of T. caries was carried out by shaking the 1000 g of wheat seeds with 2 g of teliospores in glass bottles for 10 min (Wächter et al., 2008). All test fungi and bacteria were re-isolated from artificially inoculated seeds following the surface sterilization steps and identified via microscopic examination or biochemical tests to confirm that the inoculation was successful, and the pathogens reached the internal parts of the seeds.

Ozone generation and application
Tested concentrations of O 3 were produced from atmospheric oxygen through a corona discharge type ozone generator (Pap Mobil 2000 Ozone Water Skid, Anseros, Germany) with a constant flow rate of 100 L per hour at 25°C. Anseros MP 6060 device was used to monitor the concentration of gaseous O 3 and ozonated water. A PID (Proportional integral derivative) controller was integrated into the O 3 production system to keep the O 3 concentrations at desired levels constantly. Artificially inoculated seeds or bulbs were fumigated with gaseous O 3 at the indicated concentrations in a plastic box with dimensions 39x28x14 cm. Ozonated water treatments were conducted in a tank containing ozonated water (20°C) by immersing the seeds in permeable bags. Control seeds were either treated with ambient air or tap water without O 3 .

Effects of O 3 treatment on incidence of pathogens in seeds and bulbs
The elimination success of O 3 treatments was assessed following the International Seed Testing Association (ISTA) (ISTA, 2010) validated methods. To assess the Fol and Forl inactivation after the treatment with O 3 , seeds (5 replicates of 10 seeds each plate) were placed on PDA medium and incubated at 25°C for 5 days and then the fungal growth around the seeds was monitored. Similarly, O 3 treated seeds were placed on KB or SCM medium and incubated at 28°C for 48 h to assess the inactivation of Pst, Psl and Cmm. Both fungal and bacterial colonies grown around the seeds were transferred to a new Petri Dish in every experiment and compared with the original culture and confirmed by visual inspection of colonies based on the key morphological characteristics of the microorganism. Seeds with no bacterial or fungal growth were accepted as disinfected ones (Kang et al., 2015). The assessment of the efficacy of O 3 on fungal load was performed on O 3 treated seeds and bulbs. Treated seeds and bulbs were brought to the laboratory under sterile conditions. One hundred seeds from each treatment or 10 outer/inner layers of bulbs were immersed in 100 ml of SDW and incubated on an orbital shaker for 30 min at 100 rpm. One ml of properly diluted suspension was spread on the PDA medium and the number of grown colonies after 3 days of incubation at 25°C were counted and diagnosed. The disinfection capacity of O 3 against Pcc in ornamental bulbs were assessed in storage conditions by counting the number of the decayed bulbs 36 days after treatment.

Evaluation of O 3 treatment on common bunt disease under field conditions
The efficacy of O 3 treatments on incidence of common bunt disease was evaluated under field conditions based on the methodology described by Waldow and Jahn (2007). Field experiments were performed at two different locations during 2018-2019 growing season in Izmir, Turkey: Bornova (38°27 0 05 0 ' N, 27°13 0 32 0 ' E) and Menemen (38°34 0 45 0 ' N, 27°01 0 23 0 ' E). A randomized plot design with four replicates was used at each location. Plots consisted of 1.2 by 3 m with 6 rows and the target field wheat density was 550-600 plants per plot. The number of ears infected with common bunt disease was counted in four rows in each plot and in each row one-meter length was taken. We also included hot water and fungicide treatments in the field studies to compare the efficiency of the O 3 treatments. Tebuconazole (Raxil DS 2, Bayer Crop Sciences) was applied to teliospore-contaminated wheat seeds at a dose of 150 g/100 kg seed. Seeds were immersed in a hot water at 52°C for 10 min (Waldow and Jahn, 2007). The yield (kg/plot) and 1000-grain weight (g) were also recorded in the plots remaining after marginal rows were removed.

Effects of treatment on seed germination
The phytotoxicity of the O 3 treatments on seeds was evaluated with a germination test carried out using the standard ''between paper method" described by ISTA (2010). At least 5 replications of 10 seeds were placed between the sterilized and moistened papers and incubated under conditions of 20°C for 16 h in dark and 30°C for 8 h in light. Percent of germinated seeds was recorded 10 days after treatment.

Statistical analysis
Data collected from at least two independent experiments were pooled and analyzed using the Graph Pad Prism (v.9). One-way analysis of variance (ANOVA) was used to analyze the influence of O 3 treatments on seed germination and disinfection, and the mean difference was compared using Tukey's range post hoc test, considering P < 0.05 as the threshold for statistical significance. The effects of O 3 treatments on the saprophyte load of seeds and ornamental bulbs were analyzed with Student's t-test.

Pre-soaking the seeds in water increased the efficacy of O 3 in the disinfection of seed-borne pathogens in vegetable seeds
Gaseous O 3 application for 1 h at a concentration of 5 or 10 mg O 3 /Nm 3 was effective in eliminating the saprophyte fungi on seeds which were not surface sterilized (Fig. 1A). There was a significant decrease in the number of Penicillium sp., Aspergillus sp., Mucor sp., and Rhizopus sp. colonies on the seed depending on the concentration of O 3 . Seed treatment with ozonated water at a concentration of 8 g O 3 /Nm 3 for 15, 30, and 45 min did not affect the survival of seed-borne pathogenic fungi and bacteria (Fig. 1B). Gaseous O 3 treatment at a concentration of 150 mg O 3 /Nm 3 for 90 and 120 min did not significantly affect Fol, Forl, and Cmm survival; however, it resulted in a slight reduction of Psl survival ($10 %) and a striking reduction of Pst survival (51.7 % for 90 min and 75 % for 120 min) (Fig. 1C). The germination of seeds was affected neither by ozonated water nor by gaseous O 3 treatment. Soaking the tomato and cucumber seeds in water for 12 h at room temperature followed by treatment with ozonated water or gaseous O 3 significantly enhanced the efficacy of disinfection of the seedborne bacteria and fungi (Fig. 2). Treatment of 8 g O 3 /Nm 3 ozonated water for 60 min yielded a reduction of 66.7 %, 86.7 %, 46.7 %, 43.4 % and 100 % of Cmm, Pst, Fol, Forl, and Psl in seeds, respectively, with no negative effect on seed germination rate ( Fig. 2A, B). When gaseous O 3 at a concentration of 150 mg O 3 / Nm 3 for either 90 min or 120 min was applied to seeds, all seeds were dramatically disinfected from tested seed-borne bacteria and fungi (Fig. 2C, E).

O 3 exhibited strong potential to control bacterial and fungal pathogens in ornamental bulbs
Most tulip and narcissus bulbs deposited under unfavorable conditions exhibited abundant Penicillium and Aspergillus sporulation. Three different tulips and a narcissus cultivars were directly exposed to the 75 mg O 3 /Nm 3 gaseous O 3 for 10 min and the number of Aspergillus and Penicillium isolated from outer and inner layers of bulb tissues was counted. Both Aspergillus and Penicillium growth on outer tissue of different tulip and narcissus bulbs were inhibited with a success rate of almost 100 % (Fig. 3A). However, the O 3 treatment at tested concentration was not found effective in eliminating the fungal growth on the inner tissues of bulbs N. Çetinkaya, S. Pazarlar and _ I.C. Paylan Saudi Journal of Biological Sciences 29 (2022) 103480 (Fig. 3A). We also tested the potential of ozonated water at a concentration of 8 g O 3 /Nm 3 for 60 and 120 min for controlling the bulb decay caused by the infection of Pcc. O 3 treatment for 60 min decreased the number of decayed bulbs of narcissus, hyacinth, and tulip by 95.5 %, 64 %, and 53.8 %, respectively. Furthermore, when the treatment duration was increased from 60 min to 120 min, the decrease in the decay rates of narcissus, hyacinth, and tulip increased to 99.6 %, 95.3 %, and 97.6 %, respectively (Fig. 3B, C).

O 3 treatment to contaminated wheat seeds decreased the common bunt disease in field conditions
To test whether the gaseous O 3 as a seed treatment can reduce the common bunt disease, gaseous O 3 at different concentrations (25, 50, 75 mg O 3 /Nm 3 ) was applied to T. caries contaminated wheat seeds before planting. No negative effect of the tested O 3 concentrations on the germination of seeds was found (Fig. 4A). We evaluated the incidence of common bunt disease by counting the number of infected ears in each row at the harvest. There was a significant (P < 0.05) difference in the incidence of common bunt disease between most of the treatments as compared to control (Fig. 4B, 4C). The mean value obtained for non-treated control on cv. Cumhuriyet-75 were 22.3 (±2.59) and 29.5 (±3.31) at the Bornova and Menemen locations, respectively. Ozone treatment to Cumhuriyet-75 seeds at a concentration of 25 and 50 mg O 3 / Nm 3 caused a 17.9 % (18.3 ± 1.75) and 52.6 % (10.58 ± 2.16) reduction of the number of the infected ears in Bornova, and 24.6 % (22. 2 ± 2.12) and 32.7 % (19.8 ± 1.97) reduction in Menemen, respectively. Disease incidence in non-treated control plants on cv. Gönen-98 was 34.9 (±2.47) in Bornova and 38.3 (±3.02) in Menemen. 25 and 50 mg O 3 /Nm 3 -treated plants showed less common bunt disease incidence compared to control, by 52.7 % (16.5 ± 2.0 5) and 56.3 % (15.2 ± 2.30), respectively, in Bornova, and by 36.5 % (24.3 ± 2.61) and 49.7 (19.3 ± 1.68), respectively, in Menemen. The results indicated that seed treatment with 75 mg O 3 / Nm 3 significantly reduced the number of infected plants in both tested locations and cultivars with a similar success rate of the fungicide treatment. We also evaluated the agronomical traits of wheat plants whose seeds were treated with ozone. Yield and 1000-grain weight increased significantly in both cultivars when the seeds were treated with 75 mg O 3 /Nm 3 gaseous O 3 in Bornova and Menemen locations compared to the non-treated control (Table 1).

Discussion
Disease-free seeds or bulbs are very crucial to ensure sustainable and profitable agricultural production. The limited number of studies conducted so far have shown that O 3 has potential to eliminate seed-borne pathogens. However, no comprehensive studies show the sterilization capacity of O 3 forms and concentrations in different pathogens and seeds. In the present study, we investigated the effects of gaseous O 3 and ozonated water at different concentrations on the elimination of seed-borne bacteria and fungi in vegetable seeds and ornamental bulbs. In addition, we tested the inactivation capacity of seed-transmitted common bunt fungi in wheat by O 3 treatments under field conditions. Saprophytic fungi can deteriorate seeds under unfavorable storage conditions, leading to a reduction in seed viability and can cause serious problems in human and animal health by producing mycotoxins (Rajeev and Reddy, 2017;Ramirez et al., 2018). It is also a well-known phenomenon that saprophytic microorganisms in seeds and soil can significantly influence the damage caused by primary pathogens. In this study, we show that gaseous O 3 even at relatively low concentrations is very successful in reducing the survival of saprophytic fungi, most of which are Penicillium sp. and Aspergillus sp., on tomato seeds and different ornamental bulbs including tulip and narcissus (Fig. 1A, Fig. 3A). It is not surprising that the very low concentrations of gaseous O 3 in a short time of treatment is effective to eliminate all the saprophytes which mostly live on the surface of the tomato seeds. However, we observed that saprophyte colonies in inner parts of the bulbs could not be eradicated probably due to the less quantity of the ozone concentrations diffused to inner tissues. A study conducted in barley seeds demonstrated diminished growth of Aspergillus sp. when the ozonated water was applied to seeds (Spanoghe et al., 2016). The direct contact of Penicillium citrinum, Aspergillus parasiticus, and A. flavus colonies to gaseous O 3 was found effective in inhibiting their normal growth (Savi and Scussel, 2014). Both gaseous O 3 and ozonated water treatment at a concentration of 13.8 mg l À1 and 1.7 mg l À1 , respectively, for 15 and 30 min, completely inhibited saprophytic fungi including A. flavus, A. niger, A. parasiticus, Byssochlamys fulva, Cladosporium cladosporioides, Mucor hiemalis, M. plumbeus, and M. racemosus in dried figs and both treatments for 180 min resulted in $ 90-95 % reduction in aflotoksin B1 level (Zorlugenç et al., 2008). Similarly, Ciccarese et al. (2007) demonstrated that Aspergillus spp. and Penicillium spp. were successfully eliminated in wheat, barley and pea seeds treated with gaseous O 3 at a concentration of 3 % by weight for 3 min.
Since the natural infection rate of tomato and cucumber seeds with Cmm, Pst, Fol, and Psl was not found high enough to investigate the disinfection capability of O 3 , we used artificially inoculated seeds. Ozonated water treatment did not affect the presence of any seed-borne fungi or bacteria tested, while gaseous O 3 caused a substantial and slight reduction in Pst and Psl, respectively, in seeds (Fig. 1B, C). One of the primary findings of this study was that the killing capacity of both ozonated water and gaseous O 3 is dramatically increased when the seeds were pre-soaked in water. When the seeds were soaked in water for 12 h, ozonated water treatment resulted in different levels of inactivation of fungal and bacterial pathogens depending on microbial species and duration of application, and gaseous O 3 treatment drastically eliminated all tested seed-borne fungi and bacteria from tomato and cucumber seeds without reducing the seed germination ( Fig. 2A-2E). As a technique has been applied for a long while, soaking the seeds in the water for a certain period of time depending on the plant species weaken the seed coats. The increased efficiency of sterilization in water-soaked seeds could be due to enhanced diffusion of O 3 to internal parts of seeds where the pathogens might be located. The hyphae and preincubated spores are known to be more sensitive to environmental stresses than spores (Levitz et al., 1986;Murdoch et al., 2013). The better performance of O 3 sterilization in water-soaked seeds than dry seeds may also be due to the cellular activation of fungal spores and bacteria by increased humidity which might result in O 3 sensitivity. Similarly, a better performance of fungal sterilization by microwave treatment in water-soaked seeds was reported (Mangwende et al.,  2020; Szopiń ska and Dorna, 2021; Cavalcante and Muchovej, 1993). The mechanism behind the antimicrobial properties of O 3 is attributed to either its strong oxidizing capacity or reactive oxygen species (ROS) generated during the decomposition (Bocci et al., 2009). Glycoproteins and lipids located in the bacterial and fungal cell membranes are destroyed directly by O 3 (Cho et al., 2010). Sulfhydryl groups of both apoplastic and intra-cellular enzymes are disrupted by O 3 and ROS (Menzel, 1971). O 3 also impairs the integrity of nucleic acids in living cells (Mustafa, 1990). The outermost layer of microorganism cells and their compositions are one of the primary criteria that determine the level of sensitivity to sanitizers including O 3 (McDonnell and Russell, 2001). The difference in the level of inactivation between pathogens in O 3 -treated seeds could be due to the anatomical or physiological difference between species such as thickness and composition of cell walls or antioxidant capacity of bacteria or fungi involved in scavenging of ROS. In our study, we showed that Gram negative Pst presence in non-soaked tomato seeds decreased following the treatment of gaseous O 3 while Gram positive Cmm was not affected (Fig. 1C). This difference might be due to thicker layer of peptidoglycan, which provides physical strength to the bacterial cell wall, in Gram-positive Cmm than Gram-negative Pst. Several other reports also demonstrated that Gram-negative bacteria are more sensitive O 3 than Gram-positive ones (Restaino et al., 1995;Moore et al., 2000;Zuma et al., 2009;Rangel et al., 2022). Similarly, the fact that O 3 in gaseous form did not show any effect against fungal pathogens Fol and Forl in non-soaked tomato seeds at the tested duration and concentration may be due to the thicker cell wall structure of the fungi. The resistance of fungi or yeast cells to O 3 has also been shown higher than bacteria cells and viruses (Dyas et al., 1983;Aguayo et al., 2006;de Alencar et al., 2012;Wen et al., 2020). The localization of seed-borne pathogen on seeds, could be either on testa or internal parts of seeds such as endosperm and embryo, closely affect the disinfection success of seed treatments (Koch and Roberts, 2014). However, since we used seeds that were artificially inoculated by using the infiltration technique, the efficacy difference between pathogens is not due to the localization of the pathogens.
One of the other main findings of this study is clearly that gaseous O 3 is much more effective than ozonated water at tested concentrations in eliminating seed-borne pathogens (Figs. 1, 2). We tested the maximum concentration of ozonated water as we could reach under our experimental conditions, which is substantially lower than the tested gaseous O 3 concentration. The high efficacy of gaseous O 3 than ozonated water was probably due to the difference of tested concentrations. Nevertheless, it should be noted that gaseous O 3 has almost fourfold greater diffusibility, uniform distribution and penetration ability than ozonated water, resulting in stronger disinfectant ability in most cases (Sapers, 2001;Cullen et al., 2009).
Pcc is a well-described Gram-negative bacterium known to cause soft rot in various plants including ornamentals such as hyacinth, tulip, and narcissus. Pcc can cause a dramatic loss under favorable conditions if it is not controlled properly (Boyraz et al., 2006;Van Doorn et al., 2008). The promising data found for vegetable seeds motivated us to test whether the ozonated water is effective in disinfecting the bulbs from Pcc. Since the bulbs are highly susceptible to gaseous O 3 (unprovided data), we only tested the ozonated water at a concentration of 8 mg O 3 /Nm 3 for 60 or 120 min. It was demonstrated that Pcc could effectively be disinfected from bulbs by ozonated water treatment (Fig. 3B). The direct toxicological effect of ozonated water against Pcc (formerly Erwinia carotovora subsp. carotovora) and its potential in decreasing leaf symptoms in Chinese cabbage were shown lately (Guo and Wang, 2017).
Common bunt, caused by T. caries, is one the important seedborne disease of wheat in many parts of the world (Gaudet and Puchalski, 1989;McNeil et al., 2004). T. caries is mainly managed by synthetic fungicides applied to seeds before planting (Waldow and Jahn, 2007;Matanguihan et al., 2011). Even if the number of infected seeds is very low, seed treatment is highly recommended since the teliospores in infected seeds can easily contaminate the healthy seeds during harvest and storage, and small number of teliospores have strong potential to cause disease. Tebuconazole has been widely used for seed treatment (Hoffmann and Waldher, 1981). In our study, we demonstrated under field conditions that treatment with O 3 before sowing significantly reduced the number of the common bunt infected spikes and increased the agronomical traits of wheat, with an efficacy rate much better than hot water treatment and almost like systemic fungicide Tebuconazole.

Conclusion
Synthetic fungicides and disinfectants have been used for seed treatment intensively for several decades to control seed-borne fungi and bacteria. In addition to the negative consequences of synthetic chemicals on human and environmental health, the increasing demand for sustainable and less risky food in recent years has led researchers to seek innovative and sustainable agricultural techniques for efficient production and disease control. Ozone sterilization is an environmentally friendly, effective, relatively cheap, and rapid-acting technique. We showed in this study that O 3 treatment can potentially replace the fungicide treatments. However, since it is not a selective sanitizer, it carries the risk of damaging non-target biological material such as seeds or bulbs like our case. It should be noted that the reaction of the target seed and the pathogen to O 3 should be examined very well, and the application conditions should be optimized precisely for successful O 3 sterilization.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.