Introduction

Fungal pathogens of the genus Fusarium are causal agents of many diseases not only on cereals. They are responsible for yield losses and moreover for production of mycotoxins that are harmful to human and animal health. Mycotoxic contamination of crops can occur in the field, during transport and storage (Steyn 1971). One of the most damaging fungal diseases of cereals is Fusarium head blight (FHB) caused by Fusarium culmorum, F. graminearum, F. poae, F. sporotrichioides, etc. These species are producers of Fusarium mycotoxins type B trichothecenes (deoxynivalenol, nivalenol, etc.) as well as type A trichothecenes (HT-2 toxin, T-2 toxin, etc.), and further by enniatins, beauvericin, and by zearalenone (Munkvold 2017).

Several strategies are used to prevent Fusarium colonisation of plants, and fungicide treatment is an essential part of Fusarium control management. However, the number of active ingredients in fungicides is being progressively reduced in line with the EU Biodiversity Strategy for 2030 (https://environment.ec.europa.eu/strategy/biodiversity-strategy-2030_en). Therefore, alternative seed treatments are being sought. Simple methods such as seed sorting and sieving can be used to remove infected seeds or various physical methods exploiting irradiation, ultrasound, cold plasma, or a pulsed electric field (PEF) (Stranska et al. 2023).

The pulsed electric field is a new innovative technology with huge potential in food processing, medical treatment, biomedical engineering, bioelectrochemistry, vaccination, agriculture, and many other fields too. PEF is mostly used for inactivation and elimination of microbial pathogens (fungi, bacteria, yeasts, etc.). The PEF technology works by applying a high electrical potential, in the form of pulses, across a conductive matrix (Stranska et al. 2023). In PEF processing, the product is placed between two electrodes and then submitted to the application of a high voltage short pulses of high electric fields (usually, bipolar pulses) with a very short duration to avoid heating and formation of unwanted compounds (ranging from micro to milliseconds), and field strength (1–80 kV/cm) (Zhu 2018). Irreversible electroporation of microbial membranes caused by PEF is an effective way to inactivate vegetative cells of microorganisms (Raso et al. 2014). This technology was described in German Patent 1237541 issued in 1960 (Graham 2003). It was observed that PEF treatment damages the starch surface, reduces the relative crystallinity, alters the starch in vitro digestibility (increase in the level of rapidly digestible starch and reduction in the level of slowly digestible starch), and improves the stability of emulsions (Maniglia et al. 2020). Although PEF treatment is considered a non-thermal process, in fact, the sample is heated due to ohmic heating (Han et al. 2012). For this reason, the temperature of the starch suspension must be carefully controlled to avoid thermal effects (Maniglia et al. 2021).

Zhong et al. (2019) studied the effect of PEF on Fusarium oxysporum, well-known soilborne plant pathogen. PEF deactivated most Fusarium oxysporum in nutrient solution within a few seconds. The maximum disinfection efficiency of 99.84% was obtained under the treatment conditions: 4.7 kV/cm, treatment time 60 s, and pulse width 10 μs. Evrendilek et al. (2019) describe the effect of PEF treatment on Fusarium graminearum in grain samples of wheat, barley, cabbage, and other vegetables. The PEF disinfection (12–18 kV/cm, number of pulses 200, and pulse width 1.2 μs) of the winter barley seeds decreased F. graminearum by 26.67%. The treated winter barley seeds exhibited better and faster growth and better root development.

Stranska et al. (2023) focussed on characterising the effect of PEF on mycotoxins present in malting barley. The degradation potential of PEF treatment was assessed for 16 common and emerging Fusarium and Alternaria mycotoxins. The total reduction of trichothecenes, zearalenone, enniatins, beauvericin, and tentoxin induced by PEF was up to 31, 48, 84, 36, and 46%, respectively.

The aim of this research was to study the effect of PEF on spore viability of four Fusarium species. The PEF process was tested for future use as a new innovative technology for alternative seed treatment, potentially able to kill or remove the fungi from the surface.

Materials and methods

Fungal samples

Four isolates of four Fusarium species (F. culmorum, F. graminearum, F. poae, and F. sporotrichioides), causal agents of FHB, have been chosen for the experiments in PEF. All isolates are deposited in the culture collection of microorganisms of Crop Research Institute in Prague. One isolate of every species was tested and all isolates originated from the Czech Republic (Table 1). Spore suspensions (105 spores/ml) of Fusarium isolates were prepared according to the method described by Šíp and Stuchlíková (2000). The tested isolates were grown on sterile wheat grains at 20 °C for about 2 weeks. They were then dried at 25 °C for 3–5 days. The spore suspension was prepared by releasing the conidia from the grain into water. For each experiment, 3 L of spore suspension was prepared from tap water because distilled water is not conductive.

Table 1 Origin of Fusarium isolates tested
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PEF treatment

The spore suspensions of four Fusarium species were treated in continuous PEF system (OMNIPEF; VITAVE, Czech Republic). The PEF system settings were adapted to the properties of the individual suspensions. The following parameters were constantly set for all samples: voltage of 10 kV/cm, flow rate 0.3 l/min, bipolar pulses, and pulse width 5 μs. Different frequencies (450–900 Hz) were tested on all isolates to determine the maximum frequency applicable to a particular isolate (Table 1). The spore suspensions of F. graminearum and F. poae isolates probably produced more pigments, and it was not possible to set the frequency higher than 550 (or 600) Hz, because of high conductivity of sample. The treatment itself is carried out in such a way that the spore suspension flows through the device and it is submitted to the application of a high voltage short bipolar pulses of high electric fields. After treatment, the suspension is drained from the device and is collected in sterile tubes for further study. Between each treatment, the device is always sterilised using a special product suitable for the system.

Viability test

The spore suspension passing through the PEF system was collected after the end of the process, subsequently pipetted onto Petri dishes with potato dextrose agar (PDA—Himedia), and spread with a sterile glass stick. The antibiotic gentamycin (2 ml/l) was added to PDA to suppress bacterial growth. Before applying the suspension to the PDA, the spore concentration was unified for all samples. Every Petri dish (60 mm) was inoculated by 20 µl of spore suspension and cultivated at 20 °C in the dark. All variants including untreated control were processed in ten replicates. After 3 days of cultivation, the number of colony forming units (CFU) per dish was counted. We assume that the number of CFU is equal to the number of surviving spores after PEF treatment.

Statistical analyses

The data were analysed using STATISTICA (14.0.0.15) software separately for each Fusarium species. A statistical comparison of the response of each isolate was made for a frequency of 450 Hz, which was applied uniformly to all pathogens. A general factorial ANOVA (Analysis of Variance) was performed at 95% confidence interval and 5% level of significance. When the p-value was less than 0.05, the Fisher’s least significant difference (LSD) test for multiple comparisons was carried out. The significantly different mean values were represented by different letters.

Results

All tested isolates of four Fusarium species (F. culmorum, F. graminearum, F. poae, and F. sporotrichioides) showed statistically significant differences in their response to PEF treatment (10 kV/cm, bipolar pulses, pulse width 5 μs, 450 Hz, and flow rate 0.3 l/min). Table 2 shows the results of the ANOVA with two factors (fungus and variant control/PEF), both of which were statistically significant, as well as their interactions. There were more CFU on Petri dishes with untreated spore suspensions than on the dishes with PEF treated suspensions for all isolates. Homogeneous groups show statistically significant differences between control and PEF variants for each species. The difference is statistically significant for all pathogens, but not for F. culmorum.

Table 2 ANOVA results for CFU (colony forming units) per dish for four pathogens after PEF treatment (frequency 450 Hz, 10 kV/cm, electrodes 5 mm, pulse width 5 μs, and flow rate 0.3 l/min)
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As the frequency increased from 450 to 900 Hz, the number of viable spores gradually decreased for all Fusarium species tested (Figs. 1 and 2). The isolates differed in the maximum frequency that could be set on PEF system. The highest frequency 900 Hz was used for treatment of F. culmorum and F. sporotrichioides. F. sporotrichioides conidia were completely destroyed by the 900 Hz frequency. Even at the maximum frequency of 900 Hz used for F. culmorum, 3.56% of spores survived. For F. graminearum spores, the maximum frequency was 600 Hz, but 3.21% of them survived. On the other hand, the maximum frequency of 550 Hz killed all F. poae spores. Fusarium poae responded already at the lowest frequency of 450 Hz with a strong reduction in spore viability (only 9.41% of spores survived), see Fig. 3. At the same frequency, 15.32% of F. graminearum spores, 28.91% of F. sporotrichioides spores, and 53.40% of F. culmorum spores remained viable. PEF treatment was found to be an effective tool, and the impact on the whole system (seeds and fungi) is currently evaluated. The tested Fusarium species produce different types of spores (Fig. 4).

Fig. 1
figure 1

Viability of Fusarium spores on PDA after PEF treatment (frequency 450–900 Hz, 10 kV/cm, bipolar pulses, pulse width 5 μs, and flow rate 0.3 l/min)

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Fig. 2
figure 2

Reaction of Fusarium species to different intensity of PEF (frequency 450–900 Hz, 10 kV/cm, bipolar pulses, pulse width 5 μs, and flow rate 0.3 l/min), ANOVA results, vertical bars = 0.95 confidence intervals

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Fig. 3
figure 3

Percentage of surviving spores after PEF treatment (frequency 450 Hz, 10 kV/cm, bipolar pulses, pulse width 5 μs, and flow rate 0.3 l/min) for F. poae (Fp), F. graminearum (Fg), F. sporotrichioides (Fs), and F. culmorum (Fc)

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Fig. 4
figure 4

a F. sporotrichioides—microconidia + mesoconidia, b F. culmorum—macroconidia, c F. poae—microconidia, d F. graminearum—macroconidia (photo J. Palicová et D. Novotný)

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Discussion

Fusarium head blight is one of the most important cereal diseases with a significant negative impact on human and animal health. The aim of this study was to determine the response of Fusarium spores to PEF treatment, one of the innovative physical methods that can make a significant contribution to reducing contamination in the food chain.

The effectiveness of spore treatment with PEF is likely to depend on, among other things, the size and shape of the spores. Fusarium culmorum and F. graminearum produce only curved multicellular macroconidia, F. poae mostly spherical unicellular microconidia, and F. sporotrichioides: macroconidia, mesoconidia, and different types of microconidia (Leslie and Summerell 2006). Fusarium poae, the only species tested that produces mostly microconidia and rarely macroconidia, was the most sensitive to PEF. More studies are needed to confirm that microconidia are always more sensitive to PEF.

Not many studies have been done on the topic of PEF versus fungi. Our results can be compared, for example, with the study by Zhong et al. (2019) on F. oxysporum, which produces macro- and microconidia. The increase of treatment time and electric field strength enhanced the disinfection efficiency, respectively. They obtained the maximum disinfection efficiency of 99.84% under the treatment conditions: 4.7 kV/cm, 60 s, 10 μs. The highest efficiency of 90.59% was achieved in our study for F. poae, at a higher PEF intensity (10 kV/cm, 450 Hz, bipolar pulses, 5 μs, and flow rate 0.3 l/min) than Zhong et al. (2019). The data are difficult to compare because the treatments were carried out in different facilities; in addition, in our case the aqueous spore suspension was treated, and in the other case, the fungus (probably spores with mycelium, it is not mentioned) was treated in nutrient solution. In general, the conductivity of nutrient solution is higher than that of tap water. Therefore, the viability of microorganisms should be higher in water than in nutrient solution at the same PEF setting.

Evrendilek et al. (2019) found that PEFs were a viable option for both disinfecting seed surfaces and improving seed vigour. The authors describe the effect of PEF treatment on Fusarium graminearum in grain samples of different crops. The PEF disinfection (12–18 kV/cm, number of pulses 200, and pulse width 1.2 μs) of the winter barley seeds decreased F. graminearum by 26.67%. We have achieved a higher efficiency of 84.68% on F. graminearum spores with the following settings: 10 kV/cm, 450 Hz, bipolar pulses, 5 μs, and flow rate 0.3 l/min.

The presence of spores and mycelia of mycotoxigenic Fusarium species in the food chain is a major problem. Stranska et al. (2023) demonstrated the ability of PEF technology to decontaminate mycotoxins from malting barley. The same Fusarium isolates were used in our study and for barley inoculation in the mycotoxin experiments (Stranska et al. 2023). Thus, it is possible to compare the effect of PEF on Fusarium mycotoxin content in grain with the effect of PEF on Fusarium spore viability. In the PEF treated samples, a decrease in all detectable mycotoxins was observed in dry matter of barley, when compared to the control. The degree of mycotoxin reduction correlated with the intensity of PEF treatment, which is consistent with our results.

Determining PEF equipment settings that suppress seed-borne pathogens while maintaining seed germination is crucial for the potential use of PEF in cereal seed protection. In the equipment used, PEF treatment takes place in the presence of water. Therefore, wet seeds would need to be dried after treatment, which is not practical. Further research into the PEF method is needed to find ways of using it in seed production. PEF could be one of the gentle, non-chemical alternatives to fungicide seed treatment in the future.

Conclusion

In this study, spore suspensions of four Fusarium species associated with FHB, a serious disease of cereals, were treated in a continuous PEF system to study the effect on pathogen spores. PEF was shown to be able to kill Fusarium spores of the species F. culmorum, F. graminearum, F. poae, and F. sporotrichioides. It can be concluded that PEF is an innovative tool that could be used to reduce Fusarium pathogens in cereals. Mycotoxins are produced not only in the field but also after harvest during seed storage, so removing pathogens from grains could significantly reduce the amount of mycotoxins entering the food chain. Optimisation of PEF settings is crucial for individual fungal species. To improve seed quality and replace fungicide seed treatment with PEF treatment, it is important to kill pathogens while maintaining seed germination. It is necessary to study other pathogenic fungal species (e.g. toxigenic Alternaria species or bunts) and the possibility of using PEF as one of the methods of plant, food, and seed protection in the future studies.