The effect of low-pressure plasma treatment of seeds on the plant resistance to pathogens and crop yields

Seeds are the carriers of biological and economic properties of the plants and to a great extent determine the harvest quantity and quality. One of the most important indicator of a seed quality is the degree of seed contamination by pathogenic microorganisms having the potential to cause diseases in both germinated seeds and developing plants. Chemical treatment is usually considered as the priority method for pre-sowing seed disinfection. Fungicides are used also at different stages of plant ontogenesis to protect plants during the growing season and to avoid yield loss. However, the irrational application of pesticides negatively impacts on the environment and human health.

In recent years, plasma‐based methods have been considered as effective eco-friendly techniques for application in agriculture and food technologies. Numerous studies have shown that low-temperature (non-thermal) plasma treatment of seeds had positive effects on seed germination and seedling development of many agricultural plants [1–6]. Long-term observations for both annual [7–9] and perennial [10–12] plants have revealed that plants grown from plasma treated seeds developed better than in control at different growth stages and showed an increase in biomass and crop yield.

Plasma treatment represents also a promising technology for pre-sowing decontamination of seeds that has been demonstrated for several agricultural crops such as wheat, rye, pea, barley, maize, soybean, etc [13–17]. Plasma treatment had a positive effect on plant disease resistance [18] and can be used to control plant diseases by inactivating fungal pathogens and up-regulating mechanisms of host plant culture resistance [19]. DBD plasma treatment of seeds was attempted to improve the resistance of wheat seed to drought stress [20]. Maria Perez-Piza et al [21] reported about a possibility to improve growth and yield of soybean plants grown under greenhouse conditions from infected seeds, treated by non-thermal plasma. However, the influence of plasma induced effects of pre-sowing seed treatment on plants health status during the growing season in field conditions have not yet been investigated.

In this paper, we studied the effect of pre-sowing low-pressure plasma treatment of seeds of maize (Zea mays L.), winter wheat (Triticum aestivum L.) and narrow-leaved lupine (Lupinus angustifolius L.) on seed germination, plant resistance to common diseases at different vegetation stages and crop yield. The choice of these agricultural crops is conditional from their strategic importance for the solution of problems that related with food security both regionally and globally level. Wheat and maize are important food crops cultivated worldwide. In Belarus, annually total acreage of winter wheat is over 500 thousand hectares. Lupines are grown as agricultural, ornamental or as pioneer plants in soil recovery. Despite of the fact that these crops occupy large areas of cultivation, the quality of seeds supplied on an industrial scale usually does not meet the required standards.

Fungal diseases are one of the most harmful factors decreasing wheat, maize and lupine yield. The most abundant causal agents of root rot of wheat are Fusarium spp. but species composition usually varies by season, culture and region. Seeds of maize are usually affected by fungi of the genus Fusarium, Penicillium and Ustilago zeae (causes the smut on maize). Seeds of lupine are affected by fungi of the genus Fusarium as well as anthracnose causing fungi Colletotrichum gloeosporioides and Kabatiella caulivora.

We have evaluated in field experiments the infection level of crops at different stages of ontogenesis, and the optimal conditions of plasma treatment ensuring its maximum biological efficiency were established for different species.

Content of some secondary metabolites (total phenolic compounds and anthocyanins) as well as proline content were measured in plant seedlings to better understand possible mechanisms of plasma effects on plant resistance to pathogens during vegetation.

2.1. Experimental set-up

Pre-sowing seed treatments were carried out in a planar geometry capacitively coupled 5.28 MHz plasma (CCP) reactor. A schematic diagram of the experimental set-up is shown in figure 1. The discharge is created between two water-cooled copper electrodes with diameter of 120 mm, placed in a stainless steel vacuum chamber with the inner volume of 0.053 m³. The distance between electrodes is 20 mm. The RF power is connected to the upper electrode and the lower electrode is grounded. The experimental setup is described in more detail in [14]. Seeds were put in the surface of an open, sterile Petri dish placed on the grounded electrode. Plasma treatments were performed in ambient air at a pressure of 200 Pa. The input power density measured from charge-voltage characteristics (Lissajous figures) was about 0.025 W cm⁻³ (supplementary figures S1 and S2 are available online at (stacks.iop.org/JPhysD/53/244001/mmedia)). Under the experimental conditions the gas temperature did not exceed $37{\ }^{\circ}{\rm C}$. Plasma parameters were as follows: the effective electron temperature T_e ≈ 2.3 eV and electron density n_e ≈ 5 × 10⁸ cm⁻³, which were close to those observed under same conditions in CCPs [22, 23].

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In all experiments, a pressure of 200 Pa (partial vacuum) was achieved before plasma ignition by pumping air from the chamber for approximately 7 min, so the influence of the vacuum treatment was also considered. The duration of plasma exposure was 2, 4, 5 and 7 min. For all experimental lots 200 seeds of each species were divided into four replicates of 50 seeds each. Untreated seeds were used as control.

2.2. Seed samples

2.3. Seed germination and microbiological tests

Microbiological analysis and germination tests under laboratory conditions were carried out according to the standard methods [24–26]. For laboratory testing of maize and winter wheat seeds, the germination method in rolls of filter paper was used [27]. The wet paper towels containing the seeds were rolled up and placed upright in a container which was held in a thermostat at $20{\ }^{\circ}{\rm C}\!\!-\!\!22{\ }^{\circ}{\rm C}$. Seeds of lupine were put in Petri dishes and grown in a climatic chamber at $20{\ }^{\circ}{\rm C}$. The germination and contamination of seeds for both maize and lupine were investigated on the 10th day and for winter wheat—on the 7th day after sowing. The seed was considered as germinated when the length of the seedling reached at least 50% seed length. Germination percentage (GP) was calculated as follows:

$$\begin{equation}GP = \,\frac{n}{N} \times 100\% \end{equation} \tag{ 1 }$$

where n—seeds germinated, N—total seeds analyzed. Length of seedlings (sprouts and roots) was also estimated.

Artificial inoculation of winter wheat seeds with Fusarium culmorum (F. culmorum) as one of the main pathogens of wheat worldwide caused root rot decease has been made to investigate germination of seeds with high level of fungal contamination. For seeds inoculation with spore suspension of F. culmorum we used a 7-days old culture of fungus on DPA. Mycelium was scraped with sterilized spatula adding sterilized distilled water. Suspension than was filtered and spores amount was calculated (in 1 ml) using Gorjaev's count chamber. In our experiment we used 1.5 × 10⁴ spores/mL. Seeds were sprayed with 50 ml spore suspension per 500 g of grain then dried at room temperature. After that inoculated seeds were treated with plasma during 5 and 10 min.

2.4. Field tests

A plot area of 25 m² was used in the field tests for each investigated species. Each seed plot was arranged in a randomized block design.

Pathogenic diseases were evaluated at different plant stages assessing the phytosanitary state of plants according to the following indices: disease development R (the degree of disease damage) and spread P of disease.

The spread of disease was evaluated as the ratio between the number of infected plants n and the total number N of investigated plants in plant lot (expressed as a percentage):

$$\begin{equation}P = \,\frac{n}{N}\,100\%. \end{equation} \tag{ 2 }$$

The disease development R was determined as follows:

$$\begin{equation}R = \,\frac{{\mathop \sum \nolimits_i \left( {{n_i} \times b} \right)}}{{N \times K}} \times 100\% \end{equation} \tag{ 3 }$$

where n_i—number of infected plants with the same level of fungal lesion, which corresponded to a mark b estimated in points (0—healthy, 1—weakly, 2—moderately, 3—deeply damaged), and K is the highest mark of disease scale (0–4).

The pieces of stems and leaves of infected plants with typical for each fungal infection signs were taken throughout the plot area for further analysis. Selected samples were washed in sterile water, cut and planted in Petri dishes on DPA with the cut side facing down on the nutrient medium. The infection was tested after 7 d of incubation.

2.5. Measurements of biochemical parameters

3.1. Laboratory test results

It was found as a result of laboratory tests that plasma seed treatment contributed to a decrease in contamination of maize seeds with Penicillium spp. by 20.7% in comparison to control (figure 2). However the Fusarium fungus was more resistant to plasma treatment and infection was even stimulated to some extent with vacuum. Plasma treatment also positively affected germination and the length of the sprout of maize although did not influence on root length. The most noticeable effect (increase in the laboratory germination to 96%) was observed for plasma treatment during 2.5 min. The maximum height of the germ (21.8 cm) and the length of the main root (24.2 cm) were observed for 2.5 min of treatment. Storage of seeds in the vacuum did not negatively affected germination of maize and biometric parameters of seedling and they were close to those in the control.

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Lupine seeds exhibited 100% germination both in control and plasma treated groups, and seed treatment did not practically change height of sprouts (figure 3(a)). The overall contamination in control lupine seeds was 100% and various grain fungi have been detected including Cladosporium sp. (76%), Alternaria (29.0%) as well as causing anthracnose fungi C. gloeosporioides and K. caulivora (2.0%). No C. gloeosporioides and K. caulivora fungi were observed on seeds after plasma treatment (figure 3(b)). Plasma treatment was effective also in suppressing Cladosporium sp. and Alternaria sp. by 67% and 28% respectively. Vacuum decreased Cladosporium sp. infection by 65% and suppressed Alternaria sp. on lupine.

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Plasma treatment of winter wheat seeds during 5 and 7 min leaded to a small decrease of seed germination but it remained close to that in control for shorter exposure duration (2 min) (figure 4). Plasma 2 and 5 min treatments as well as vacuum resulted in increase of sprouts and roots length but there was no difference between plasma 7 min and control groups (LSD = 5.0 mm). Plasma treatment reduced contamination of winter wheat seeds with Alternaria spp. and Fusarium spp. (figure 4(b)). A significant decrease in infection by Alternaria spp. achieved during 7 min of plasma exposure. Vacuum also led to reduce infection level by Fusarium spp. but was not effective against Alternaria spp. compared plasma treatment. Subsequent plasma exposure reduced Alternaria spp. on winter wheat. However at the experimental conditions Fusarium infection was negligible.

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For artificially inoculated winter wheat seeds with F. culmorum plasma treatment led to significant increase of length of sprouts (by 7.8–19.5 mm) in all experimental groups in comparison with the control (table 1). Plasma 5 min resulted in increase of roots length by 13.4 mm whereas 10 min had no significant difference compared to control.

Table 1. Influence of plasma seed treatment on morphological parameters of winter wheat seedlings from seeds inoculated with F. culmorum before treatment.

	Sprouts		Root
Treatment	length (mm)	increase (mm)	length (mm)	increase (mm)
Control	59.6	—	103.6	—
Plasma 5 min	79.1	19.5	117.0	13.4
Plasma 10 min	67.4	7.8	104.8	1.2
LSD	5.3	—	7.0	—

3.2. Field test

Boil smut is one of the most dangerous infections that affect maize crops during the growing season. The causative agent of maize smut disease is a basidiomycete fungus U. zeae. In the vegetative growth stage V3 (collar of 3rd leaf visible) we performed artificial inoculation of maize plants with 0.2% suspension of U. zeae teliospores to establish the efficacy of plasma treatment in suppressing smut pathogen in the field conditions. Although the environmental conditions did not contribute to intensive development of boil smut in maize crops in V9 stage (9th leaf visible), nevertheless the infection level of plants from the treated seeds was 3 times less than in control (figure 5(a)).

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One of the most harmful diseases at the first stages of lupine ontogenesis is root rot. At the stages of 3rd-4th leaves emerged, disease development and root rot spread in control plants reached to 47.8% and 65% correspondingly, while these indicators in plants from the treated seeds for 5 min did not exceed 6.9% and 16.2% (figure 5(b)).

At the later stages, the most harmful disease of lupine is anthracnose spread by infected seed and rain-splash of spores from infected plants. Field experiments showed that pre-sowing plasma treatment of seeds significantly reduced anthracnose on lupine and suppressed the disease development right up to the flowering stage (figure 5(c)). However, even at a later stage (green bean), the infection level in plants from treated seeds for 7 min remained significantly lower (by 20%) than in control group.

In field experiments winter wheat root rot severity at BBCH 25 (tillering) and BBCH 32 (two nodes) stages in control was 12.9% and 16.2% respectively (figure 6). The disease severity in plants from treated seeds for 5 min was 8.8% and 10.0% correspondingly. In this experiment, a part of non-treated and plasma treated winter wheat seeds were treated additionally with seed dressing chemical Maxim Forte, SC (2.0 L t⁻¹) and sown in one experimental plot together with control and plasma treated seeds to compare the efficacy of physical and chemical methods against root rot disease. Finally chemical seed dressing was the most effective (6.8% disease severity in BBCH 32). No synergistic effect of both methods was found.

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In general, pre-sowing plasma treatment of seeds resulted in improving the structure of the yield (table 2).

Table 2. Effect of plasma seeds treatment on crop yield.

			Yield
	Treatment	Mass of 1000 grains (g)	center/hectare	Increase in yield (%)
Maize	Control	189.4	94.4	—
	Plasma 4, 5 min	191.0	96.0	1.7
	LSD	—	1.0
Lupine	Control	133.4	17.5	—
	Plasma 4 min	144.2	22.2	26.8
	LSD	—	4.7	—
Winter wheat	Control	48.9	68.8	—
	Plasma 5 min	49.6	70.4	2.3
	Plasma 5 min + Maxim Forte, SC	50.3	71.7	4.2
	Maxim Forte, SC	50.4	72.0	4.7
	LSD	—	1.9

At present, the research data concerning the field experiments on effect of plasma seed treatment on growth and productivity of agricultural plant are very limited. Jiang et al [17] showed the efficiency of cold helium plasma treatment in improving the growth and physiological level of wheat leading to increased yield in field experiments. It was observed in greenhouse experiments that cold plasma seed treatment had the potential to control tomato bacterial wilt [18], and could improve soybean plant dry weight, plant height, stem diameter and yield of plants grown even from infected seeds [21], and the effects persisted throughout the entire cycle of plants. At the same time authors claimed that field studies need to be addressed in order to confirm these results.

We investigated for the first time the effectiveness of plasma seed treatment in reducing a number of fungal crop diseases such as boil smut of maize, root rot of lupine and winter wheat at various stages of plant growth under natural field conditions.

Plasma treatment contributed to reduce infection level of boil smut in maize at V9 stage. In the early stages of lupine growth (3rd–4th leaves emerged), spread and development of root rot in plants from treated seeds were 53% and 41.8% less than in control respectively (figure 5). Plasma treatment suppressed anthracnose on lupine up to the flowering stage and the infection level remained much lower than in control even at green bean stage. The anthracnose fungus is seed-borne and infected seed is the main source of inoculum dispersal and disease outbreaks [34]. Thus, the observed absence of the anthracnose spread in lupine crops can be associated with a high fungicidal effect of pre-sowing plasma treatment against anthracnose causing fungi K. caulivora and C. gloeosporioides (figure 3).

Even for artificially inoculated winter wheat seeds with fungus F. culmorum which is one of the major stressor limiting wheat plant growth and productivity, the treatment for 5 min led to significant increase of length of sprouts and roots by 19.5 and 13.4 mm, respectively in comparison with control (table 1). In field experiment plasma treatment (5 min) reduced winter wheat root rot severity in BBCH 32 stage but was less effective than chemical seed dresser Maxim Forte, SC (figure 6).

It was shown that due to a decrease in the level of seed infection, stimulation of field germination, early seedling growth and plant resistance to pathogens during the vegetation period, the winter wheat grain yield increased by 2.3%, maize—by 1.7%, narrow-leaved lupine—by 26.8% compared to control plants (table 2). In the case of winter wheat, the use of both plasma and chemical treatment of seeds contributed to a decrease in plant infection, which led to an increase in yield, although the yield in the variant with chemical treatment was 2 times higher than in the case of plasma treatment.

Since secondary metabolites such as phenols, etc play an important role in plant defense systems, the biochemical parameters of plants associated with the formation of general non-specific resistance of plants to abiotic and biotic stresses has been studied. For instance, high concentrations of phenols can increase plant resistance to stress [35, 36]. Free proline accumulation in plant cells indicates enhanced adaptation of plants to various stress conditions [37]. Measurements were made on maize seedlings (as a model object) because of high knowledge of this culture, rapid germination and accumulation of biomass seedlings, sensitivity and a clear response to various stresses.

Plasma seeds treatment for 2 and 4 min resulted in a significant increase in activity of non-enzymatic antioxidants such as phenols and anthocyanins in roots of 7-days maize seedling (figure 7). The content of proline also significantly increased in seedling roots but decreased in seedling leaves in comparison to control plants. In leaves, only 4 min treatment caused a substantial accumulation of anthocyanins.

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Plasma treatment also positively influenced biomass of maize although reduced length roots with increased exposure time until 4 min (figure 7(d)). In roots, plasma exposure caused a significant increase in activity of non-enzymatic antioxidants which could be an important part of the plants defense system against biotic and abiotic stresses [38, 39].

The accumulation of proline to high levels in plant cells under stress could greatly increase the reactive oxygen species (ROS) scavenging capacity of said cells and reduce the potential for oxidative damage. Proline could potentially acting as storage reserve of carbon and nitrogen, a compatible osmolyte, a buffer for cytosolic pH, a scavenger of ROS as well as an aid to balancing cellular redox status [37].

Plasma exposure for 5 min caused a significant increase in total phenolic content in winter wheat leaves—by 21.1% and 15.5% at BBCH 25 and BBCH 32 stages correspondingly (figure 8). Amount of phenolics increased for plasma 5 min (by 15.5%) and for combined treatment 'plasma 5 min + fungicide' (by 7.0%).

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It was reported in [40] that the important first line in plant defense against infection is provided by the very rapid synthesis of phenolics and their polymerization in the cell wall. We asumed that the accumulation of phenolic compounds in plants in response to plasma seed treatment could be the reason for the reduction of plant disease during vegetation.

It is likely that plasma treatment of seeds affects the plant organism as an inducer, causing sensitization of the plant's defense mechanisms, a kind of 'priming', which provides the formation of stress-tolerant plants with an increased 'red-ox' status, as evidenced by an increase in the content of non-enzymatic antioxidants in the tissues. It is believed that the inducers that cause priming do not directly induce defense reactions, but create the prerequisites for the activation of resistance mechanisms that are realized upon subsequent contact of the body with stressors [41]. The use of inducers is in many cases accompanied by stimulation of plant growth, does not cause the development of resistance by pathogens, and, moreover, can positively affect crop productivity and product quality.

The results obtained under laboratory and field conditions have demonstrated high efficiency of low-pressure radio frequency plasma in stimulation of the germination and early growth of seeds, in the reduction of their infection with pathogenic fungi which resulted in the improving of sowing value, viability, health status and crop yield of some important agricultural plants: maize, narrow-leaved lupine and winter wheat.

Pre-sowing plasma treatment reduced fungal contamination of seeds, and had a long-lasting effect under natural field conditions decreasing or suppressing of fungal diseases of plants during the phase of active vegetative growth (in lupine—up to the flowering stage) as well as led to improve the yield of plants.

Plasma treatment enhanced the resistance of crop plants via promoting the accumulation of non-enzymatic antioxidants and proline in plants.

So, application of plasma technology in seed treatment can reduce the use of agrochemicals during the crop cycle or in some cases, eliminate the chemicals at the pre-sowing stage.

This work is partially supported by the State Committee on Science and Technology of the Republic of Belarus (SCST) and the Belarusian Republican Foudation for Fundamental Research (BRFFR) under Grand Nos. F19LITG-008 and B19LATG-003.