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Article

Pre-Sowing Seed Treatments with Cold Atmospheric Plasma for the Control of Seedling Blights of Winter Wheat

by
Tzenko Vatchev
1,
Ivo Todorov Yanashkov
1,
Plamena Marinova
2,3,* and
Evgenia Benova
3
1
Plant Protection Department, Institute of Soil Science Agrotechnologies and Plant Protection, Agricultural Academy, Panayot Volov Str., Kostinbrod, 2230 Sofia, Bulgaria
2
Faculty of Forest Industry, University of Forestry, 10 Kliment Ohridski Blvd., 1797 Sofia, Bulgaria
3
Clean & Circle Center of Competence, Sofia University, 15 Tzar Osvoboditel Blvd., 1504 Sofia, Bulgaria
*
Author to whom correspondence should be addressed.
Processes 2025, 13(3), 632; https://doi.org/10.3390/pr13030632
Submission received: 20 December 2024 / Revised: 17 February 2025 / Accepted: 19 February 2025 / Published: 23 February 2025
(This article belongs to the Section Chemical Processes and Systems)
Browse Figure
Versions Notes

Abstract

A wide range of seed-borne and soil-borne plant pathogens belonging to various fungal and fungal-like species cause pre-emergence seed decay and post-emergence seedling blights of wheat and other small-grain cereal crops. To prevent the death of the seedlings, poor establishment and reduced stand of the crops, extensive crop rotations, planting good-quality seeds and seed treatments with fungicides are used on regular basis. This study is aimed at assessing the efficacy of pre-sowing seed treatments with cold atmospheric plasma for the disinfestation of winter wheat seed from economically important fungal and fungal-like pathogens. Uninoculated or surface-inoculated with Fusarium culmorum, Bipolaris sorokiniana or Pythium ultimum wheat seeds, the cultivar Madara was treated by cold plasma produced either by microwave torch (MW) or underwater diaphragm discharge (UW) with low power at very short treatment times, or remained untreated controls. As per the treatments, the seeds were sown in a ready-to-use growing medium comprising a mixture of light and dark moss peat (w:w) 90–95%, 5–10% perlite and 3–5 kg/m3 CaCO3, having an electrical conductivity of 40 mS/m, pH (H2O) of 5.5–6.5 and moisture content of 60–70%, filling in 250 × 250 × 70 mm aluminum flat seed trays (40 grains per tray, four trays per treatment). The plants were cultivated for 45 days in a growth chamber held at (20 ± 2) °C, set to a cycle of 8 h/night and 16 h/day under fluorescent light of 2000–3000 lux intensity. For each replicate, disease incidence (DI) was determined as the total percentage of missing, dead and apparently symptomatic plants. Seed treatment with a microwave plasma torch with a power of 16 W for 40 s significantly (p < 0.001) reduced seedling blights caused by F. culmorum, B. sorokiniana and P. ultimum by 46.8%, 51.0% and 77.3%, respectively, but limited the emergence of wheat seedlings by 15.9% on average. Simultaneously, the effectiveness of underwater discharge seed treatments reached an average of about a 60% reduction of seedling blight caused by F. culmorum and B. sorokiniana and about 37% of the disease caused by P. ultimum. Pre-sowing treatments with a MW plasma torch with an input power of 11 W and treatment time of 60, 90 or 120 s exposure also showed significant (p < 0.001) effects in controlling winter wheat seedling blights caused by the three pathogens. The effectiveness of the treatment increased with increasing the time period of exposure and reached full disease control (>80% reduction) for B. sorokiniana and P. ultimum seedling blights. This study demonstrated that pre-sowing treatment with a microwave plasma torch and underwater diaphragm discharge at a relatively low input power and short exposure time can be used for disinfestation and the effective control of seedling blights in winter wheat caused by seed-borne fungal pathogens, such as Fusarium culmorum and Bipolaris sorokiniana, and fungal-like oomycetes, such as Pythium ultimum.

1. Introduction

A wide range of primary pathogen species relating to different soil-inhabiting taxonomic groups, including fungi and fungal-like oomycetes, cause seedling blight or pre- and post-emergence damping-off in winter wheat and other small-grain cereal species. This results in the death of seedlings, poor establishment and reduced stand of the crop, stunting, reduced biomass, plant vigor and, eventually, reduced grain yield per plant and unit area [1,2,3,4,5]. The infection of wheat in the early growth stages with pathogens belonging to the genera Fusarium, Bipolaris and Pythium can result in dramatic yield losses ranging from 36% to 80% [2,6,7,8,9]. Later, during the stem elongation period and the emergence of inflorescence, infection can develop into foot and root rot, stalk rot and many other disease symptoms. Thus, Fusarium spp. is the causal agent of seedling blight, common root rot, foot rot, stalk rot, ear and head blight and other diseases of cereal crops. Among the genus Fusarium, F. culmorum (W.G. Smith) Sacc. has proven to be the most aggressive and widespread pathogenic species in winter wheat in Bulgaria [10,11]. Bipolaris spp., represented by the most commonly identified species in the country, B. sorokiniana (Sacc.) Shoem. (anamorph), syn. Helminthosporium sativum Pammel, C.M. King & Bakke, Cochliobolus sativus (Ito & Kurib.) and Drechsler ex Dastur (teleomorph), is associated with seedling blight, common root rot, node cankers and spot blotch on the leaves of wheat and other small-grain cereals [10,12]. Pythium ultimum Trow is one of the various Pythium spp. species identified in the roots of cereal crops causing severe damage to wheat [1,2,13]. Under conditions of prolonged waterlogging, the pathogen can cause serious pre-emergence seed decay, post-emergence damping-off, symptoms of stunting and the severe root rot of plants [14,15].
High infection levels of seedling blight diseases may also occur where seed is sown in cold seedbeds and in regions with heavy, poorly drained soils. Generally, the causative species are widespread and can survive in soil for many years [16,17,18]. The disease can also be seed-borne. The seed-borne inoculum sticks to the seed coat or presents as concomitant contamination with the seed. It is considered a major source of infection and can be a point source for introduction into new locations [19,20,21].
The seed-borne and soil-borne diseases of wheat and other cereals are controlled primarily by planting good-quality seeds, seed treatment with fungicides and crop rotation. Seed dressing and less frequent seed coating, seed encrusting or pelleting, providing varying degrees of coverage with fungicides and other ingredients, are widely used and may be effective for controlling pre-emergence and post-emergence diseases of grains [22]. A disadvantage of fungicide seed treatment is that it can secure only a limited period of protection, usually confined to the earlier growth stages of plants. Another issue is that only a few seed-dressing fungicides have broad-spectrum activity to target a variety of diverse genera of plant pathogens, and even products containing two or more active substances have limited efficacy. In addition, there is a significant unmet necessity for the post-harvest treatment of cereals referring to the disinfection of harvested grain as a preparative operation for safe storage. Due to the generation of reactive oxygen and nitrogen, cold plasma technology offers an alternative to ionizing radiation (e.g., X-rays or γ-rays) and ozone treatment for the surface decontamination of microorganisms, detoxification of mycotoxins and use of pesticides in the post-harvest processing [23,24].
Plasma is a complex system with a variety of active components: electrically charged particles (electrons and ions); chemically active excited atoms, molecules and radicals; UV radiation; and heat components. Nonthermal (non-equilibrium) plasma at an atmospheric pressure usually has an electron temperature Te of about 1–2 eV, while the heavy particle temperature (so-called gas temperature, Tg) can vary from room temperature to above 1000 K. When the gas temperature is low enough (below 45 °C), the heat component can be eliminated and the plasma is called cold plasma. This is of great importance for the plasma treatment of biological systems since the heat component produces the main damage. Electrical discharges operating at atmospheric pressure usually produce plasma with high temperatures, which is not applicable for bio-medical and agriculture applications. To avoid thermal damage, plasma is produced in a pulse regime or the treated samples are placed at some distance from the active plasma in the afterglow region [25]. In the latest case, only long-living active particles can reach the samples and produce a chemical reaction.
Another widely used approach is treatment with plasma-activated water (PAW)—a liquid treated by plasma for producing reactive species in it—and after that, treating it with biological samples. The composition of plasma-activated water and the concentration of reactive oxygen and nitrogen species (RONS) obtained with two plasma systems, a microwave plasma torch (MW) and underwater diaphragm discharge (UW), have been extensively studied in ref. [26].
The microwave plasma torch operating at an optimized discharge condition (argon as working gas, low input power) demonstrates its ability to produce cold atmospheric plasma (CAP) in the active plasma-sustaining region [27,28] and can be used for the treatment of thermosensitive materials and biological systems like seeds [29]. The active plasma region is also rich in short-living particles and more chemical reactions are possible in this case.
The plasma can also be produced directly to the liquid media, as is the case of underwater discharge, often also called diaphragm discharge [30,31]. This kind of discharge is promising, especially for agriculture applications since the samples (seeds, plants) are treated directly in water, which decreases the possible damage [28,32]. The optical emission spectrum of the UW discharge in the range of 200–1000 nm was previously discussed in refs. [28,30]. The water temperature changes during the treatment in the underwater discharge cameras were studied in ref. [29] as well as the effect of the plasma treatment on the seeds of three Bulgarian durum wheat cultivars treated with cold plasma in different variants.
Cold atmospheric plasma has proven its applicability in the treatment of biological systems in recent years. With the development of new plasma sources that allow for the plasma to be at a low temperature and not damage living organisms, tissues and plants, plasma applications in areas such as medicine, ecology and agronomy have also emerged. After detailed research and proof of the bactericidal properties of plasma treatment, new areas for plasma applications are revealed. The possibility of cold plasma being applied to seed treatment in order to increase germination, improve sprout development and increase resistance to diseases and stress is of particular interest. Evaluating the impact of cold plasma on seedling growth properties, seed germination has been the focus of numerous research studies [33,34]. Recent studies have evaluated various cold plasma devices with different parameters and their effects on wheat seeds [35]. Depending on the properties of the plasma devices, both positive and inhibitive effects have been observed. Motrescu [36] assessed the effect of voltage conditions to find the optimum conditions for seed treatments and showed that even a single plasma device working in different operational conditions can lead to both inhibitory and promotion effects on plant development. Only a few of the processing conditions proved to stimulate the growth and production of some nutraceutical compounds [36]. The key factor in applying plasma treatment to seeds is the optimization of plasma sources so that promising results are obtained and negative impacts are avoided. The extensive work to date on the influence of different plasma sources on seed properties after plasma treatment has led to identifying the appropriate combinations of the plasma source and duration of treatment, positively affecting seed germination and seedling growth in durum wheat cultivars [37,38,39,40,41]. A detailed investigation was made of how the treatment with two plasma sources, microwave plasma torch (MW) and underwater diaphragm discharge (UW), affects the germination, germination energy, morphological parameters of plants, yield and osmoregulation [29]. This study proves that the treatment of conventional wheat seeds of different varieties in appropriate operational regimes of these two plasma sources does not damage them and does not decrease their germination energy, shoot length and root length. Even more, at the optimized operational condition of the plasma sources (electrical power and duration of treatment), positive effects on seed germination and seedling growth in the three studied durum wheat cultivars are obtained.
The application of CAP for the inactivation of microorganisms and viruses and the effect of the active components in plasma and plasma-activated water are presented in ref. [42] prove the effective decontamination of surfaces and liquids. The effect is based on the interaction of the microorganisms with the active particles in the plasma, which are strong oxidizers.
As a logical continuation of our previous works, this present study is aimed at demonstrating the capabilities of non-thermal atmospheric plasma for seed disinfection and disinfestation. The possibility of using cold plasma to treat seeds for the inactivation of various pathogens is of special interest [43,44,45].
Our previous studies have found a positive effect of cold plasma as a pre-planting seed treatment technique of Fusarium-infected wheat on germination rate and plant growth [46].
The plasma sources used in this present investigation are the same as in ref. [29]: microwave plasma torch (MW) and underwater diaphragm discharge (UW). The main advantage is that their characteristics and the optimal operating conditions for seed treatment, ensuring treatment without damage and with positive effects, have already been established. As a next step, the possible disinfecting effect of plasma in the treatment of infected seeds is investigated. This study is aimed at assessing the efficacy of pre-sowing seed treatment with a microwave plasma torch and underwater diaphragm with low power and very short treatment times for the disinfestation of winter wheat seed from fungal and fungal-like pathogens.

2. Materials and Methods

2.1. Experimental Pathosystems

Three different plant pathosystems representing different pathogen classes are chosen and considered individually as model systems in this work. These include winter wheat as a host component and either F. culmorum or B. sorokiniana as a pathogen component. Both are generally the most aggressive of their respective genera and are widespread fungal pathogens in cereal crops. Pythium ultimum is used as a representative of the fungal-like oomycetes. Although soil-inhabiting Pythium ultimum is not considered a seed-transmitted pathogen, wheat seeds artificially infested with this along with the other two pathosystems were used to evaluate the ability of cold plasma to surface-disinfest cereal seeds from fungal and fungal-like pathogens and thus managing the economically important diseases they cause.

2.2. Inoculum Production and Inoculation Technique

The pathogenic species involved in this study were previously isolated from diseased winter wheat plants cultivated in the field. Their pathogenicity is confirmed by soil infestation tests. Reisolates, morphologically identical to the original inoculants, are obtained from symptomatic test plants, stored on Potato Dextrose Agar (PDA) slants at 4 °C and used during the studies. Inoculum of the three pathogens is produced after 7 days of pure-culture cultivation in 90 mm Petri plates, each containing 10 mL of Potato Dextrose Agar (PDA) and incubated in the dark at 26 °C. The content of 10 Petri plates (agar medium with mycelium and spores) of each pathogen is mixed in 1000 mL of sterile distilled water (SDW) in a household mixer at medium speed for about 30 s or until a uniform suspension is obtained. An amount of 250 mL of mycelium and spore suspension of each pathogen species is sprayed on 1 kg of winter wheat seed cultivar Madara and mixed thoroughly for 3 min. Seeds from the same batch are sprayed with pure agar medium suspended in SDW in an identical manner and used as an uninoculated control in all experiments. The inoculated seeds and uninoculated controls are stored for a short period (overnight) in a refrigerator at 4 °C or used within 1 h for treatment procedures.

2.3. Plasma Treatment of Seeds

Plasma treatment is performed with two plasma devices: microwave plasma torch and underwater diaphragm discharge, which are presented in detail in ref. [29] and their characteristics are shown in Table 1. As shown in ref. [28], the temperature of the samples treated by MW torch (controlled by infrared camera and contact thermometer) increases with the electrical power and the treatment time. Keeping the power below 20 W, we decrease the wave power while increasing the treatment time in order to provide a seed temperature below 45 ⁰C. The treatment discharge conditions are as follows:
(1)
Microwave plasma torch with input power 16 W and treatment time 40 s.
(2)
Microwave plasma torch with input power 11 W and treatment time 60 s.
(3)
Microwave plasma torch with input power 11 W treatment time 90 s and 11 W for 120 s with ice cooling.
(4)
Underwater diaphragm discharge treatment in the container with applied voltage of 5 kV electrode, denoted by “+”, or grounded electrode, denoted by “−”.
Both plasma systems are compared in Table 1.
The microwave plasma torch is operated at 2.45 GHz frequency produced by Sairem Surfatron wave launcher in argon gas flowing inside a quartz discharge tube [46,47,48,49]. Geometrical configuration of the discharge tube with outer diameter of 7 mm and inner diameter of 3 mm at an Ar (99.99999%) gas flow of 5 L/min is used. The Surfatron is connected to a solid-state microwave generator (GMS 200 W, SAIREM–FRANCE, Décines-Charpieu, France); this configuration of the microwave plasma torch allows for obtaining cold plasma with temperature close to the room temperature. For seed treatment, the active plasma-sustaining region in the plasma torch is used in such a way that all plasma components and short-living particles can react with the sample. The low input power of the wave (11–16 W) makes the MW plasma torch very energetically effective.
The underwater diaphragm discharge used in this study was constructed and developed at the Faculty of Chemistry, Brno University of Technology [29,30]. This device produces plasma directly in the water. The camber for the plasma water treatment is divided into two containers with 50 mL volume each with respect to the two planar, high-voltage (HV) electrodes and separated by a dielectric membrane (D). The power supply is produced by a Lifetech s.r.o., Brno, Czech Republic. The high-frequency (15 kHz) voltage of 5 kV is applied to the electrode denoted by “+”. The electrode denoted by “−” is grounded.
The evaluated treatment regimes are presented in Table 2. They were chosen on the base of previous investigations on the plasma properties at given discharge conditions and the plasma treatment effects on the seeds [26,27,28,29,30,31,32]. The treatment at higher microwave power (16 W) can increase the temperature of the seeds, and in that case, the treatment time is the shortest (40 s) for minimizing the thermal damage. The microwave power in the three variants is as low as possible (11 W) and the effect of the treatment time is investigated, varying it (60 s, 90 s, 120 s). The treatment time by UW discharge is fixed at 5 min. Each of the containers is filled with 50 mL water. One of the samples is put in the container with the powered electrode denoted as “+” and the other sample is in the container with the grounded electrode, denoted as “−”.
Each of the treated samples contains 200 wheat grains placed in a 90 mm sterile Petri plate.
Identically manipulated inoculated and non-inoculated seeds, without plasma treatment, serve as controls for all runs of the experiments. Treated and untreated seeds are stored in a refrigerator at 4 °C for no more than 24 h and then used for sowing in the bioassays/experiments conducted in the study.

2.4. Sowing and Cultivation

All biological tests are conducted in flat aluminum plant (seed) trays (250 × 250 × 70 mm) with holes. Each seed tray is filled with 4 L of fresh ready-to-use growing medium comprising a mixture of light and dark moss peat (w:w) 90–95%, 5–10% perlite and 3–5 kg/m3 CaCO3, having an electrical conductivity of 40 mS/m, pH (H2O) of 5.5–6.5 and moisture content of 60–70% (Fitogea, Sofia, Bulgaria). Grains in the untreated controls and cold plasma-treated groups are seeded in five rows per tray with eight grains per row (row × plant spacing of 4 × 3 cm) at 3 cm depth and completely covered with growing mixture (40 grains per tray, Figure 1). Four seed trays (experimental units) are used for each treatment. After planting, prepared experimental units are placed in completely randomized blocks in growth chamber held at (20 ± 2) °C and set to a cycle of 8 h/night and 16 h/day under fluorescent light of 2000–3000 lux intensity at the surface of the growing medium. The latter is watered two to three times a week according to the plants’ needs. In all experimental runs, plants are grown for 45 days.

2.5. Type, Time and Frequency of Assessment

Three successive assessments were performed at 10, 30 and 45 days after planting. At the first and second assessments, all emerged seedlings with apparently healthy aboveground parts were counted in each seed tray. At the third assessment (day 45) all remaining seedlings were uprooted and carefully examined for chlorosis, stunting, foot and root rot and damping-off symptoms. For each replicate, disease incidence (DI) was determined as the total percentage of missing, dead and symptomatic plants. To confirm that the disease symptoms were due to the same pathogen inoculated on wheat seeds, isolations from symptomatic plant roots and basal stems collected at random from each treatment at the end of the experiments were routinely performed on water agar (WA) acidified PDA and oatmeal agar (OA) media. Additionally, seedlings from non-inoculated, plasma-treated seeds were examined for the presence of any phytotoxic effects by reference to the untreated, non-inoculated controls.
The effectiveness (E%), i.e., the level of disease control of each cold plasma seed treatment x pathogen combination was calculated on the basis of disease incidence values using Abbott’s formula [50], as follows:
E% = [1 − (X/Y)] × 100,
where X corresponds to the disease incidence in treated seedlings and Y corresponds to the disease incidence in the untreated control plants infested with the same pathogen species.

2.6. Statistical Analyses

Each of the experiments was repeated over two successive years. The independent repetitions yielded similar results. Separate analyses of each trial showed homogeneous variance of the experimental error between repetitions, and therefore, the data from the repeated trials were combined for analysis. The resulting data sets were processed by analysis of variance using F-test for significance of the means of treatments and Least Significant Difference (LSD) values were computed at p ≤ 0.05 [51]. Where applicable, treatment means were separated by Duncan’s multiple range test (p ≤ 0.05, [52]). All analyses were performed with IBM SPSS Statistics version 19.0 for Windows.

3. Results

In all experiments, pre-plant seed treatment with cold plasma significantly (p < 0.001) reduced seedling blight attacks caused by each of the three target pathogens and substantially improved wheat stands derived from pathogen-infested seed, as shown in Table 3 and Table 4. Plants that developed from plantings of the untreated pathogen-infested seed were severely affected by the disease, showing poor stands due to seedling blight losses. As a result, the highest disease incidences were generally recorded in the untreated control stands reaching between 76% and 89% seedling loss caused by the individual pathogen species. Soon after emergence, some of the seedlings that had survived pre-emergence damping-off began to exhibit chlorosis and foot and root rot and also died. Seedling loss continued until days 15–30 after planting in almost all plant trays but still varied significantly (p < 0.001) between treatments.
The pre-sowing treatment of wheat seed with a microwave plasma torch with power 16 W for 40 s (Table 3) effectively reduced disease incidence in stands developed from seed contaminated with F. culmorum or B. sorokiniana to 46.7% and 37.5%, respectively (Table 3). As compared to the untreated controls, the effectiveness of the treatment for each of these two pathogens was 46.8% and 51.0%, respectively. As compared to the other cold plasma treatments in this experiment, pre-sowing seed disinfestation with the plasma torch treatment at the discharge conditions chosen gave the highest disease control of Pythium seedling blight, decreasing the disease incidence by 77.3%. Notably, the application of 16 W for 40 s limited the emergence of wheat seedlings by 15.9% on average from the two repetitions of the experiment. This is connected with the increase of the temperature of the plasma torch, which produces some thermal damage. In order to avoid this, in the next experiments, the microwave power was reduced to 11 W and the treatment time was increased. In addition, the dependence on the treatment time was studied for the 11 W power by varying it (60 s, 90 s, 120 s). Therefore, in all subsequent experiments of this study, the wheat grains were exposed to lower power of MW plasma for an extended time period (Table 3). The seed treatments either with UW+ for 5 min or UW− for 5 min had similar effects on the disease caused by each of the three pathogens. About 30% to 34% disease incidence was recorded in wheat stands developed from seed infested with F. culmorum or B. sorokiniana after treatment, while both disinfestation regimes led to 47.7% and 47.5% seedling loss in Pythium-infested plants. As compared to the untreated controls, the effectiveness of the two seed treatments reached an average of about 60% reduction of seedling blight caused by F. culmorum or B. sorokiniana and about 37% by P. ultimum.
Pre-treatments with a MW plasma torch with an input power of 11 W and treatment time of 60, 90 or 120 s exposure also showed significant (p < 0.001) effects in controlling winter wheat seedling blights caused by seed-borne F. culmorum, B. sorokiniana or P. ultimum compared to the untreated control groups (Table 4). The effectiveness recorded in stands from seed preliminarily subjected to cold plasma disinfestation increased substantially when prolonging the exposure period from 60 to 120 s. The recorded disease control rates reached a full level of control—more than an 80% disease reduction for B. sorokiniana and P. ultimum and a 73% reduction of seedling loss caused by F. culmorum. In all undertaken experimental runs, all plants deriving from uninoculated seed pre-treated with cold plasma in the abovementioned treatment regimens emerged without delay and further developed normally as compared to those developed from their untreated counterparts.

4. Discussion

In this present study, pre-sowing cold plasma treatment of pathogen-contaminated winter wheat seed showed significant effects in controlling seed-borne seedling blights caused by F. culmorum, B. sorokiniana and P. ultimum. Seed treatment with a MW plasma torch with a power 16 W for 40 s developed a significantly higher percentage of successfully emerged and disease-free seedlings. This treatment regimen exceeded the controlling effect of underwater discharge in a “+” chamber with a treatment time of 5 min or underwater discharge in a “−” chamber for 5 min in reducing seedling blight of wheat caused by P. ultimum. However, the treatment with 16 W was shown to result in a substantial reduction (by 15.9%) in wheat stands, which were not artificially inoculated with a plant pathogen. From the experimental study, we can conclude that this reduction is due to some thermal damage at a microwave power of 16 W. Therefore, to avoid this undesired and unacceptable effect of plasma treatment on the target crop, all further tests in this present study were performed with a lower power of the MW torch, e.g., 11 W, following the earlier obtained dependence between the microwave power and the plasma temperature [28]. The decrease in the power requires an increase in the treatment time to reach similar results. Applying MW plasma treatment of pathogen-infested seed at a power of 11 W was as effective or even superior to other seed treatments in controlling the three pathogen-induced diseases. Increasing the exposure time from 60 to 90 and 120 s significantly decreased the seedling loss and correspondingly increased the level of disease control of the seedling blights caused by the three pathogen species.
From the findings of this work, it was revealed that treatment by underwater discharge for 5 min in a “+” chamber or by underwater discharge for 5 min in a “−” chamber substantially confined F. culmorum and B. sorokiniana seedling blights and foot and root rots by approximately 55% to 65% compared to the untreated counterparts. The comparison of the results between the two plasma sources shows better performance of the MW torch at a longer treatment time of 120 s.
Two aspects of the effects of plasma seed treatment on the protection of cultivars from pathogens have been discussed so far in various studies. There is evidence that plasma treatment leads to an increase in the resistance of treated seeds to pathogens. On the other hand, the ability of plasma to inactivate various groups of microorganisms has long been proven. The role of plasma treatment in fungal inactivation is less known in comparison to the effect on bacteria. Plasma consists of many different active components, all of them having an effect on biological objects. UV emitted from the plasma affects the enzyme activity of cells. Reactive oxygen and nitrogen species also have a strong oxidative effect on the cell membranes and charged particles rupture the cell membranes [43,45]. The effect of plasma treatment on relative enzyme activities in cells has been proven and the pronounced antimicrobial effect can be used for surface sterilization [42]. When applying plasma technologies for the treatment of biological objects, two approaches are most commonly used—direct treatment and indirect treatment. Direct treatment allows for the object to be in direct interaction with the active part of the plasma where significant UV radiation and plasma particles are present. The indirect plasma treatment, for example, in liquid, is characterized by lower UV radiation since UV is absorbed by the medium but higher concentrations of active species transfer to the liquid, such as O3, NO, OH and Nox, and transformed to long-lived H2O2, NO2− and NO3−. These two treatment regimens differ significantly in what active particles produced by the plasma interact with the treated object. The working gas strongly affects the active particles created and the effects of the plasma treatment. A higher concentration of RONS can be produced when the working gas is nitrogen with an oxygen admixture but also at a higher plasma temperature [53,54]. The RONS can also be produced during the interaction of the plasma in an inert gas (argon in our case) with the ambient air. In argon plasma, the gas temperature is much lower, and therefore, for biological systems, the use of argon plasma is more suitable. In this case, when the plasma working gas is argon, the available nitrogen and oxygen particles obtained by the interaction with plasma particles and air are highly desirable for biological applications. The changes in the reactive particles that are also present when the plasma is in contact with or surrounded by water have been widely studied due to the inevitable presence of water in the treatment of biological objects. Unfortunately, it is now proven that the medium surrounding the plasma not only changes the composition and concentration of the reactive particles that come into contact with the treated object but also affects the plasma parameters [55]. After detailed studies of the processes at the plasma–air and plasma–water interface, the effects of the presence of different objects on this boundary during plasma treatment have remained unclear. This work is particularly interesting for possible applications, as it allows us to compare the effect of two plasma sources—one suitable for a dry processing mode without the presence of water and one with plasma created directly in water. In these two cases, the type and concentrations of the RONS are significantly different depending on whether the plasma interacts with air or water as the surrounding medium.
Many factors can lead to significant changes in the physical and chemical properties of plasma. Undoubtedly, the most significant is the type of plasma source, but no less important to study are the variations in discharge conditions and the changes in interaction with different types of objects. The two plasma sources used in this study were selected to allow for both of the abovementioned methods for the plasma treatment of seeds. The microwave plasma torch was used in the mode of direct interaction with the seeds and, accordingly, the direct interaction with UV, electromagnetic fields, high concentrations of electrons and argon ions, as well as short-lived active species. The treatment with underwater discharge, where the interaction is indirect through water, is characterized by significantly higher concentrations of ROS and RNS and a much more acidic environment. An additional investigation of the mechanisms of plasma treatment effects is necessary in the future. Nevertheless, the selected plasma sources demonstrated their ability for significant seed decontamination and increased efficiency at optimized operational conditions.
In general, the concentrations of RONS in PAW are linear dependent on the time of treatment. The UW discharge treatment of water produces a significantly higher concentration of H2O2 compared to the MW discharge. The typical concentrations for H2O2 for UW and short treatment times are between 10 to 30 mg/L and below 5 mg/L for the MW. In contrast, the MW device is able to produce NO2− and NO3− with concentrations much higher than the UW. NO2− concentrations in PAW for the UW discharge are up to 0.5 mg/L for treatment times of less than 10 min and up to 3 mg/L for the MW plasma torch. In addition, the pH of the PAW from the underwater discharge is significantly lower than that obtained from treatment with a MW torch [26].
All active plasma components—charged particles (electrons and ions), UV radiation and short- and long-lived reactive particles like RONS—synergistically act on the treated samples when the treatment is conducted by the active plasma sustaining region as it is in this work. This allows for obtaining better effects in a short treatment time In comparison to chemical treatment, the ozonation process or other methods that use only one of the active plasma components [26], the plasma treatment is more effective and more efficient due to the sinergetic effect of all active plasma components.
In the frame of this study, the MW plasma system operational regime is optimized for controlling the seed-borne seedling blights caused by F. culmorum, B. sorokiniana and P. ultimum in winter wheat seed. Additional research is currently underway to optimize the seed-treatment regimens using CAP against the aforementioned pathogenic species along with other seed-borne fungal pathogens, like smuts and bunts, in winter wheat and other small-grain crops. However, further studies will be needed to permit the commercial use of this alternative to the chemical methods of pre-plant seed treatment.

5. Conclusions

This study demonstrates that the pre-sowing application of cold atmospheric plasma can be used for seed disinfestation and the effective control of seed-borne diseases in winter wheat caused by two fungal pathogens, Fusarium culmorum and Bipolaris sorokiniana, and one fungal-like oomycete, Pythium ultimum. As compared to the underwater diaphragm source, the microwave torch at low power of 11 W and longer treatment time of 120 s gives better performance in reducing seedling blights caused by the three pathogen species. While being effective in controlling the diseases, the pre-sowing treatment of wheat seed with a microwave plasma torch with a power of 16 W for 40 s limited the emergence of wheat seedlings probably due to some thermal damage to the seed. These results point to future technology for pre-sowing treatment using a MW plasma torch at low power (11 W) and a treatment time of about 120 s.

Author Contributions

The investigation concept was proposed during personal discussion between all authors. Plasma source operation and plasma treatment, E.B. and P.M.; plant growing and analysis of the results, I.T.Y. and T.V.; writing—original draft preparation, E.B., I.T.Y., T.V. and P.M.; writing—review and editing, E.B., I.T.Y., T.V. and P.M. All authors have read and agreed to the published version of the manuscript.

Funding

This paper was supported by the Bulgarian National Science Fund under Grant No. KΠ-06-KOCT/16, national co-financing for the participation of Bulgarian collectives in established actions under the European Cooperation in Science and Technology Action COST. This work was supported by the European Cooperation in Science and Technology Action CA19110—Plasma applications for smart and sustainable agriculture (PlAgri). This research was partially supported by by the Project “Clean Technologies for Sustainable Environment—Water, Waste, Energy for a Circular Economy”, financed by the Bulgaria Programme “Research, Innovation, and Digitalisation for Smart Transformation”: BG16RFPR002-1.014.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Acknowledgments

The authors would like to thank Frantisek Krcma from the Brno University of Technology for the provision of the underwater diaphragm discharge equipment for our laboratory.

Conflicts of Interest

The authors declare no conflicts of interest.

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Processes 13 00632 g001
Figure 1. Sowing and cultivation procedure.
Figure 1. Sowing and cultivation procedure.
Processes 13 00632 g001
Table 1. Plasma source parameters.
Table 1. Plasma source parameters.
Devices Microwave Plasma TorchUnderwater Diaphragm Discharge
Processes 13 00632 i001Processes 13 00632 i002
Plasma systemsSairem Surfatron wave launcher, quartz discharge tubePolycarbonate container divided into two parts
Power supply typeSolid-state microwave generator (GMS 200 W, Sairem)Lifetech DC power supply
Input power11 W, 16 W50 W
Voltage 5 kV
Frequency2.45 GHz wave frequency15 kHz
Type of treatmentDry treatment/In waterIn water
Treatment volumeVariableTwo fixed chambers of 50 mL each
Treatment times40, 60, 90 and 120 s5 min
Table 2. Evaluated CAP seed-treatment regimes.
Table 2. Evaluated CAP seed-treatment regimes.
VariantDevice Input Power/VoltageTreatment Time
MW 16_40Microwave plasma torch16 W40 s
MW 11_60Microwave plasma torch11 W60 s
MW 11_90Microwave plasma torch11 W90 s
MW 11_120Microwave plasma torch11 W120 s
UW+Underwater diaphragm discharge“+” 5 kV electrode5 min
UW−Underwater diaphragm discharge“−” grounded electrode5 min
Table 3. Effects of cold plasma seed treatment regimens (MW_16 and UW) on the incidence of seedling blight and root rot of winter wheat caused by three plant-pathogen species.
Table 3. Effects of cold plasma seed treatment regimens (MW_16 and UW) on the incidence of seedling blight and root rot of winter wheat caused by three plant-pathogen species.
Seed Contamination/Infestation
TreatmentF. culmorumB. sorokinianaP. ultimum
DI (%) aE (%) cDI (%)E (%)DI (%)E (%)
Untreated control87.7 a b-76.5 a-75.8 a-
MW 16_4046.7 b46.837.5 b51.017.2 c77.3
UW+32.5 c62.930.0 c60.847.7 b37.1
UW–30.8 c64.934.2 b55.347.5 b37.3
F =
P =
SE =
LSD0.05 =		288.95
0.00018
1.55724
4.59387		 248.4
0.00034
1.36575
4.02897		 171.99
0.00020
1.82688
5.38931
a For each treatment, the disease incidence index (DI) was expressed as the percentage of diseased (dead or symptomatic) wheat plants 45 days after seeding. b Values of disease incidence index within the column followed by different letters are significantly different at p ≤ 0.05 according to Duncan’s multiple range test. c The effectiveness (E, level of disease control) of each treatment was calculated on the base of the disease index value using Abbott’s formula.
Table 4. Effects of cold plasma seed treatment regimens (MW_11) on the incidence of seedling blight and root rot of winter wheat caused by three plant-pathogen species.
Table 4. Effects of cold plasma seed treatment regimens (MW_11) on the incidence of seedling blight and root rot of winter wheat caused by three plant-pathogen species.
Seed Contamination/Infestation
TreatmentF. culmorumB. sorokinianaP. ultimum
DI (%) aE (%) cDI (%)E (%)DI (%)E (%)
Untreated control88.9 a b-79.1 a-79.1 a-
MW 11_6048.5 b45.445.3 b42.750.0 b36.8
MW 11_9049.6 b47.222.3 c71.826.4 c66.6
MW 11_12023.8 c73.210.3 d87.014.3 d81.9
F =
P =
SE =
LSD0.05 =		349.62
0.000008
1.43963
4.17046		 119.13
0.000003
2.77696
8.04454		 201.93
0.000002
3.03787
3.53553
a For each treatment, the disease incidence index (DI) was expressed as the percentage of diseased (dead or symptomatic) wheat plants 45 days after seeding. b Values of disease incidence index within the column followed by different letters are significantly different at p ≤ 0.05 according to Duncan’s multiple range test. c The effectiveness (E, level of disease control) of each treatment was calculated on the base of the disease index value using Abbott’s formula.
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Vatchev, T.; Yanashkov, I.T.; Marinova, P.; Benova, E. Pre-Sowing Seed Treatments with Cold Atmospheric Plasma for the Control of Seedling Blights of Winter Wheat. Processes 2025, 13, 632. https://doi.org/10.3390/pr13030632

AMA Style

Vatchev T, Yanashkov IT, Marinova P, Benova E. Pre-Sowing Seed Treatments with Cold Atmospheric Plasma for the Control of Seedling Blights of Winter Wheat. Processes. 2025; 13(3):632. https://doi.org/10.3390/pr13030632

Chicago/Turabian Style

Vatchev, Tzenko, Ivo Todorov Yanashkov, Plamena Marinova, and Evgenia Benova. 2025. "Pre-Sowing Seed Treatments with Cold Atmospheric Plasma for the Control of Seedling Blights of Winter Wheat" Processes 13, no. 3: 632. https://doi.org/10.3390/pr13030632

APA Style

Vatchev, T., Yanashkov, I. T., Marinova, P., & Benova, E. (2025). Pre-Sowing Seed Treatments with Cold Atmospheric Plasma for the Control of Seedling Blights of Winter Wheat. Processes, 13(3), 632. https://doi.org/10.3390/pr13030632

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