Non-Thermal Plasma Can Be Used in Disinfection of Scots Pine ( Pinus sylvestris L.) Seeds Infected with Fusarium oxysporum

: The aim of this study was to use di ﬀ use coplanar surface barrier discharge (DCSBD) non-thermal plasma for the disinfection of pine seed surfaces infected with Fusarium oxysporum spores. Artiﬁcially infected seeds of Scots pine ( Pinus sylvestris L.) were treated with plasma for the following exposure times: 1 s, 3 s, 5 s, 10 s, 15 s, 20 s, 30 s, and 60 s, and subsequently germinated on agar medium in Petri dishes at room temperature for the estimation of seed germination and disinfection e ﬀ ect of plasma treatment. Results of the treated samples were compared to the control samples, which were prepared as follows: seeds uninfected and non-treated with plasma (ﬁrst control); seeds infected with F. oxysporum and non-treated with plasma (second control); and seeds infected with F. oxysporum , non-treated with plasma, but sterilized with 30% perhydrol (third control). Obtained results indicate that 3 s plasma treatment was an optimal time to inhibit F. oxysporum growth, and at the same time, increase the seed germination. In addition, our results are the ﬁrst to show the practical application of non-thermal plasma in disinfecting infected Scots pine seeds and improving their germination. According to the results of this study, non-thermal plasma can serve as a seed surface disinfectant in the regeneration of di ﬀ erent pine species.


Introduction
Scots pine (Scotch pine, Pinus sylvestris L.) is the most common conifer of the Pinaceae family and gymnosperms growing in the Northern Hemisphere. It is a valuable and commercially important

Seed Inoculation
Scots pine seeds, before inoculation with F. oxysporum spores, were surface disinfected in 30% H 2 O 2 for 20 min. Subsequently, seeds were rinsed six times in sterile distilled water and aseptically air dried for 1 h [29]. Aliquots (100 µL) of water after the sixth rinse were aseptically spread onto R2A (Becton Dickinson) and PDA (Potato Dextrose Agar, Becton Dickinson, NJ, USA) to confirm surface sterilization of seeds from bacteria and fungi, respectively. Both the preparation of spore suspension and the seed inoculation were performed following the procedure described by Evira-Recuenco [30], with minor modifications. Briefly, the F. oxysporum spore suspension was prepared by washing a 7-day-old culture growing on PDA (Potato Dextrose Agar, Becton Dickinson, NJ, USA) at 28 • C with sterile saline solution. Fungal spore concentration was counted using a hemocytometer, diluted to a concentration of 10 6 spores/mL and then sonicated. Seeds were inoculated with the fungal spore suspension in 50 mL centrifuge tubes and incubated at 28 • C in gentle shaking conditions for 20 min. The seeds were separated from solution and air dried in a laminar hood overnight.
Both the surface sterilized seeds and the ones treated with sterile saline solution were used as the negative control (Control 1). Surface sterilized seeds inoculated with fungal spores were used as the positive control (Control 2). Additionally, seeds inoculated with fungal spores were treated with 30% H 2 O 2 for 20 min (Control 3) to compare the effectiveness of chemical and non-thermal plasma disinfection methods.

Description of Diffuse Coplanar Surface Barrier Discharge (DCSBD)
Diffuse coplanar surface barrier discharge (DCSBD), as a special type of the dielectric barrier discharge, generates low-temperature plasma at atmospheric pressure in a thin layer on the dielectric surface [31]. Configuration of the DCSBD plasma with the experimental setup of the plasma treatment is shown in Figure 1. Many parallel electrodes situated at the bottom side of the ceramic Al 2 O 3 plate and cooled by the oil are powered by a high voltage sinusoidal signal. Alternating-current (AC) signal with an amplitude of approximately 20 kV peak to peak with a frequency of 14 kHz was generated by the HV generator LIFETECH VF 700 (Lifetech, Czech Republic). Plasma created in a thin layer is composed of many micro-discharges moving on the ceramic surface along the electrodes. Electrodes are not in contact with the plasma, which prolongs their life span.

Seed Cultivation
Infected seeds were treated with plasma and directly germinated onto a surface of Yeast extract Peptone Dextrose (YPD) agar (Becton Dickinson, NJ, USA), a solid medium suitable for the cultivation of fungi, in sterile Petri dishes. Thirty seeds were aseptically placed on the surface of the dish with YPD agar. The seed germination was run under controlled growing conditions (28 °C, in darkness for 12 days).
During the experiment, some characteristics were observed [15,25]: 1. disinfection efficiency (DE): percentage of seed without mold spreading around seeds, 2. seed germination (SG): percentage of germinated seeds (germination is positive if a 1 mm radicle occurs), 3. germination rate (GR): the percentage ratio of germinated seeds at the beginning (4 th day) to germinated seeds at the end (12 th day) of the seed germination, 4. germination index (GI): the total number of germinated seeds to the respective day (4 th , 7 th , 9 th , and 12 th day) of germination.

Statistical Analysis
The obtained data were analyzed using the STATISTICA software (Statistica 13, StatSoft Inc. Tulsa, OK, USA) at the significance level of 0.05. Logarithmic transformation (y = log(x)) of the basic data was used for normalization before the analysis. One way analysis of variance (ANOVA) was used to evaluate the influence of the plasma treatment on characteristics of seed germination and surface disinfection. The detailed testing of experimental variances from each other was done using Tukey's honest significant difference (HSD) test.

Morphological Observation
Morphological observations of seeds and seedlings after different exposure time to non-thermal plasma treatment and controls were carried out with the naked eye. The following characteristics were recorded: seed coat color, external structure of the seedlings, amount and color of cotyledons, shoots, and fine roots.

Effect of Plasma Treatment on Seed Disinfection and Germination
It was found that all plasma treatments (for different exposure times) showed relatively high values of disinfection efficiency (more than 85%) during all days of seed germination ( Figure 2). The highest disinfection efficiencies were observed after 3 s, 5 s, 15 s, 20 s, 30 s, and 60 s of plasma treatment, and statistical difference was observed from controls 1 and 3 (Table 1).

Plasma Treatment
DCSBD plasma treatment ( Figure 1) was performed at different exposure times: 1, 3, 5, 10, 15, 20, 30, and 60 s. After switching on the plasma at the applied power of 400 W, seeds were put into the plasma for the specific time and moved rotationally. Rotational movement of the seeds was carried out by the orbital shaker, which moved the whole discharge panel at the frequency of 330 rpm for the homogeneous plasma treatment of the seeds.

Seed Cultivation
Infected seeds were treated with plasma and directly germinated onto a surface of Yeast extract Peptone Dextrose (YPD) agar (Becton Dickinson, NJ, USA), a solid medium suitable for the cultivation of fungi, in a sterile Petri dishes. Thirty seeds were aseptically placed on the surface of the dish with YPD agar. The seed germination was run under controlled growing conditions (28 • C, in darkness for 12 days).
germination rate (GR): the percentage ratio of germinated seeds at the beginning (4th day) to germinated seeds at the end (12th day) of the seed germination, 4.
germination index (GI): the total number of germinated seeds to the respective day (4th, 7th, 9th, and 12th day) of germination.

Statistical Analysis
Data were analyzed using the STATISTICA software (Statistica 13, StatSoft Inc. Tulsa, OK, USA) at the significance level of 0.05. Logarithmic transformation (y = log(x)) of the basic data was used for normalization before the analysis. One way analysis of variance (ANOVA) was used to evaluate the influence of the plasma treatment on characteristics of seed germination and surface disinfection. The detailed testing of experimental variances from each other was done using Tukey's honest significant difference (HSD) test.

Morphological Observation
Morphological observations of seeds and seedlings after different exposure time to non-thermal plasma treatment and controls were carried out with the naked eye. The following characteristics were recorded: seed coat color, external structure of the seedlings, amount and color of cotyledons, shoots, and fine roots.

Effect of Plasma Treatment on Seed Disinfection and Germination
It was found that all plasma treatments (for different exposure times) showed relatively high values of disinfection efficiency (more than 85%) during all days of seed germination ( Figure 2). The highest disinfection efficiencies were observed after 3 s, 5 s, 15 s, 20 s, 30 s, and 60 s of plasma treatment with no statistical difference between these six treatments and Control 1 and 3, which were subsequently different from Control 2 ( Table 1).  Disinfection efficiency (%) of Scots pine seeds inoculated with F. oxysporum exposed to different times of non-thermal plasma treatment after the 4 th , 7 th , 9 th , and 12 th days of fungal incubation. Control 1: seeds uninfected and non-treated with plasma; control 2: seeds infected with F. oxysporum and non-treated with plasma; control 3: seeds infected with F. oxysporum, non-treated with plasma, but sterilized with 30% perhydrol. More details are given in the Methods. All data including the sets of the three controls, measured at the end (12 th day) of the Disinfection efficiency (%) of Scots pine seeds inoculated with F. oxysporum exposed to different times of non-thermal plasma treatment after the 4th, 7th, 9th, and 12th days of fungal incubation. Control 1: seeds uninfected and non-treated with plasma; control 2: seeds infected with F. oxysporum and non-treated with plasma; control 3: seeds infected with F. oxysporum, non-treated with plasma, but sterilized with 30% perhydrol. Table 1. Effect of diffuse coplanar surface barrier discharge (DCSBD) plasma treatment on Scots pine seeds infected with F. oxysporum. Characteristics measured (mean and standard deviation (SD) are given) at the end of experimental cultivation (12th day). Tukey's HSD (Honest Significant Difference) test was used. Significant differences at p < 0.05 are indicated by different letters. Control 1, control 2, and control 3, see Figure 2. All data including the sets of the three controls, measured at the end (12th day) of the experiment, are summarized in Table 1. The highest seed germination was observed when seeds were treated with plasma for 3 s on all measurement days while the lowest with plasma treatment for 60 s (Figure 3). The highest seed germination rate (GR) after seven days of cultivation was found when seeds were treated with plasma for 3 s. In contrast, the highest GR after nine and 12 days of cultivation was observed for seeds treated with plasma for 10 s in both cases (Figure 4). The germination index calculated for the end of the cultivation period (12th day) was highest when seeds were treated with plasma for 3 s ( Table 1). The sterilization effectiveness was at the level of 95.3% on the 4th while on the 7th, 9th, and 12th day of seed cultivation, the level of sterilization effectiveness was at 94.0%. A total of 14.0% of the seeds germinated on the fourth day, while 86.0% germinated on the seventh, and 96.7% germinated on the twelfth day of seed cultivation ( Figure 5).

Mean
Forests 2020, 11, x FOR PEER REVIEW 6 of 11 germination index calculated for the end of the cultivation period (12 th day) was highest when seeds were treated with plasma for 3 s ( Table 1). The sterilization effectiveness was at the level of 95.3% on the 4 th while on the 7 th , 9 th , and 12 th day of seed cultivation, the level of sterilization effectiveness was at 94.0%. A total of 14.0% of the seeds germinated on the fourth day, while 86.0% germinated on the seventh, and 96.7% germinated on the twelfth day of seed cultivation ( Figure 5).    . Germination rate of Scots pine seeds exposed to different times of non-thermal plasma treatment after the 4 th , 7 th , 9 th , and 12 th days of seed germination. Control 1, control 2, and control 3, see Figure 2. More details are given in the Methods. The percentage of Scots pine seed germination exposed to different times of non-thermal plasma treatment after the 4th, 7th, 9th, and 12th days of seed germination. Control 1, control 2, and control 3, see Figure 2.
Forests 2020, 11, x FOR PEER REVIEW 6 of 11 germination index calculated for the end of the cultivation period (12 th day) was highest when seeds were treated with plasma for 3 s ( Table 1). The sterilization effectiveness was at the level of 95.3% on the 4 th while on the 7 th , 9 th , and 12 th day of seed cultivation, the level of sterilization effectiveness was at 94.0%. A total of 14.0% of the seeds germinated on the fourth day, while 86.0% germinated on the seventh, and 96.7% germinated on the twelfth day of seed cultivation ( Figure 5).    . Germination rate of Scots pine seeds exposed to different times of non-thermal plasma treatment after the 4 th , 7 th , 9 th , and 12 th days of seed germination. Control 1, control 2, and control 3, see Figure 2. More details are given in the Methods. . Germination rate of Scots pine seeds exposed to different times of non-thermal plasma treatment after the 4th, 7th, 9th, and 12th days of seed germination. Control 1, control 2, and control 3, see Figure 2.

Morphological Observation
Although the test exposure times of seeds to non-thermal plasma were found to be non-lethal for them, their extended germination time was observed when compared to the control ones. The use of non-thermal plasma did not affect the color change of the seed coat. The external structure of the seedlings that grew from seeds treated with non-thermal plasma did not differ from the control ones. The number of green cotyledons varied from four to six, and the shoots and fine roots were all well recognized.

Discussion
Among the microbial diseases of plants, plant pathogenic fungi are dominant causal agents leading to severe yield loss in a number of crop plants, vegetables, fruits, and in forestry [32].To date, many chemical products have been effectively used in the control of plant diseases caused by a wide-range of fungi [33]. Although universal application of broad spectrum fungicides definitely helps in controlling the fungal diseases in plants, at the same time, there have been several reports suggesting that these fungicides have been posing severe hazardous effects on the environment for several years. Moreover, extensive use of chemical fungicides has also resulted in resistance development in many plant pathogenic fungi against such fungicides [34]. Furthermore, loss in soil fertility is another major concern [35]. In this context, the search for novel, effective, and environmentally-friendly solutions for the reduction in plant pathogenic fungi is a prime area of research [36]. One such solution is the development of the seed disinfection method without the use of chemical fungicides [37].
Scientists are increasingly interested in using non-thermal plasma for the disinfection of plant seeds and bulbs [38]. The lethal effects of non-thermal plasma on fungi, viruses as well as vegetative cells and spores of bacteria have been proven by Kordas et al. [6], Terrier et al. [39], Klämpfl et al. [40], Ryu et al. [41], Tyczkowska-Sieroń and Markiewicz [42], Pignata et al. [43], and Sakudo et al. [44]. Antifungal effect of non-thermal plasma treatment against fungus, F. oxysporum on Scots pine seeds, was also proven by our studies. After 1 s of treatment with plasma, more than 80% of fungal growth was inhibited. However, so far, little research has been done on the disinfection of conifer seeds using this method. Most of the available data relates to the seeds of cereals and crops. The disinfecting effect of plasma on seeds has been confirmed in studies by many authors. Kordas and

Morphological Observation
Although the test exposure times of seeds to non-thermal plasma were found to be non-lethal for them, their extended germination time was observed when compared to the control ones. The use of non-thermal plasma did not affect the color change of the seed coat. The external structure of the seedlings that grew from seeds treated with non-thermal plasma did not differ from the control ones. The number of green cotyledons varied from four to six, and the shoots and fine roots were all well recognized.

Discussion
Among the microbial diseases of plants, plant pathogenic fungi are dominant causal agents leading to severe yield loss in a number of crop plants, vegetables, fruits, and in forestry [32]. To date, many chemical products have been effectively used in the control of plant diseases caused by a wide-range of fungi [33]. Although universal application of broad spectrum fungicides definitely helps in controlling the fungal diseases in plants, at the same time, there have been several reports suggesting that these fungicides have been posing severe hazardous effects on the environment for several years. Moreover, extensive use of chemical fungicides has also resulted in resistance development in many plant pathogenic fungi against such fungicides [34]. Furthermore, loss in soil fertility is another major concern [35]. In this context, the search for novel, effective, and environmentally-friendly solutions for the reduction in plant pathogenic fungi is a prime area of research [36]. One such solution area is the development of the seed disinfection methods without the use of chemical fungicides [37].
Scientists are increasingly interested in using non-thermal plasma for the disinfection of plant seeds and bulbs [38]. The lethal effects of non-thermal plasma on fungi, viruses as well as vegetative cells and spores of bacteria have been proven by Kordas et al. [6], Terrier et al. [39], Klämpfl et al. [40], Ryu et al. [41], Tyczkowska-Sieroń and Markiewicz [42], Pignata et al. [43], and Sakudo et al. [44]. The antifungal effects of non-thermal plasma treatment against the fungus, F. oxysporum on Scots pine seeds, was confirmed by our studies. After 1 s of treatment with plasma, more than 80% of fungal growth was inhibited. Little research has previously been done on the disinfection of conifer seeds using this method Most of the available data relates to the seeds of cereals and crops. The disinfecting effect of plasma on seeds has been confirmed in studies by many authors. Kordas and coauthors [6] studied the use of low-thermal plasma as a disinfectant method for winter wheat grain. They found that the use of this plasma for a 10 s exposure time resulted in a reduction of the seed-born fungi and positively affected the basic values determining seed lot quality as well as the development of winter wheat in the initial growth stage [6]. Other authors have evaluated antifungal plasma effect against Aspergillus sp. and Penicillum sp. using seeds of chickpeas, corn, barley, oats, wheat, and lentils, and showed that a significant reduction in the growth of the studied fungi was achieved after 15 min of exposure [45]. Zahoranová et al. [12] observed growth inhibition of Alternaria alternata, Aspergillus flavus, and F. culmorum on maize seeds after treatment with plasma while Kang et al. [46] and Ochi et al. [47] reported the inhibition of F. fujikuroi growth on rice seeds. Other authors [45,48,49] have also used non-thermal plasma for the sterilization of legumes, cereals, and nut seeds. Simultaneously, they observed that its use positively affected the seed growth and plant development.. Moreover, Go et al. [50] reported that exposure of postharvest peppers to non-thermal plasma for 90 s resulted in 50% reduction of the development of F. oxysporum. Avramidis et al. [51] observed that a 60 s exposure of F. culmorum and Ascochyta pinodella to non-thermal plasma caused cell wall and membrane damage and cytoplasmic leakage in these fungi. Non-thermal plasma destroys microorganisms by the synergistic action of reactive particles, ionized ions, electrons, and UV radiation [52,53].
The effectiveness of using non-thermal plasma as a sterilizing and plant growth promoting agent varies, depending on the gas used in the apparatus and the type of microorganisms that appear on the sterilized material [44]. However, it should also be mentioned that the prolonged plasma exposure time, which is necessary to inhibit the growth of certain pathogens, may damage the seeds and weaken their germination [12,15]. Until now, the impact of plasma on the seed germination of Scots pine has not been well studied yet. In the research of Šerá et al. [15], the time necessary to inhibit the growth of F. circinatum was one minute and caused complete inhibition of seed germination. In our research, seeds exposed to plasma for longer than 10 s had their germination lowered when compared to non-treated seeds.
For commercial use of non-thermal plasma for seed sterilization, it is necessary to develop an optimal exposure time that will provide a desirable effect without any damage to the seeds. In any case, plasma is a promising technology that will help to establish a successful strategy to regenerate trees through seeds.

Conclusions
The results of this work demonstrate the ability of using non-thermal atmospheric plasma for effective and rapid disinfection of Scots pine seeds to prevent the spread of the plant pathogen, F. oxysporum. Short exposure time to this factor caused stimulation in seed germination compared to untreated seeds. However, exposure longer than 10 s inhibited their germination. This research is the first report on the use of non-thermal plasma treatment for the disinfection of Scots pine seeds and for the estimation of seed germination and seedling growth. It seems that non-thermal plasma may be used in the regeneration of Scots pine and other woody species through seeds. To consider this method as an alternative to chemical disinfection, more research should be performed with the use of other fungal species to optimize plasma treatment conditions.