Keywords

6.1 Background

The agriculture and food industries face many challenges, including some resulting from climate change and environmental pollution. Climatic change has caused the emergence of new diseases and changes in plant susceptibility to diseases [76]. Some estimate a 5–50% reduction in crop yield as a result of climatic change by 2100 [10]. Thus, climate change, sustainable agriculture, food preservation, and improved storage of fresh produce have become important issues to be resolved. Conventional approaches to solve problems in agriculture and food processing are mostly focused on the use of chemicals. However, chemical-based techniques have frequently shown their limitations with respect to safety and emergence of resistance. Alternative technologies may be needed to overcome these challenges.

Non-thermal plasma is a promising technology to solve problems in the agriculture and food industries. Although intensive studies of plasma application to agriculture and foods started relatively later compared to medical applications, enormous advances in knowledge and technical development have been made in the last decade. Non-thermal plasma generated under atmospheric, low, or medium pressure has been examined for its potential in food sanitation, food storage, plant disinfection, and enhancement of seed germination and growth [12, 36, 106, 187, 278]. Although most investigations have been performed under laboratory conditions, the application of plasma at industrial and field scales is currently increasing. This chapter presents a synthesis of studies performed on plasma applications in agriculture and food production. We attempted to include as many studies as possible, and any omissions were unintentional.

6.2 Application of Non-thermal Atmospheric Pressure Plasma to Prevent Seed Borne Infections

6.2.1 General Treatment of Seeds

Seed is a basic and vital input for agricultural productivity considering that ninety percent of food crops are grown from seed. To guarantee health and quality (maximum germination above 80%) of seeds, they are generally subjected to pre-harvest manipulation directed towards improving germination and to deliver protection against pathogens and related pests and diseases. Factors that can impair seed quality are related to: biological factors (pathological, entomological, animal grub); physiological factors (physiological disorders, nutritional imbalances, maturity); environmental/cultural factors (e.g. climate, weather, soils, water relations, light intensity); mechanical damage during processing; extraneous matter (growing medium, vegetable matter, chemical residues); and genetic variation and aberrations [148]. The chosen seed treatment should be functional across a wide variety of soil types, cultural practices and environmental conditions. They aim at changing physical form to facilitate sowing (pelleting), enhance germination by improving physiological performance (priming) and extend longevity by removing pathogens. Commonly applied modes of seed treatment can be categorized into: (1) mechanical methods (scarification, separation from infectious agents), (2) physical methods (electron beam treatment, hot water treatment: dry heat treatment: aerated heat treatment, radiation treatment, microwave, ultrasound), (3) biological methods (treatment with beneficial microorganisms including fungi and bacteria e.g. species of Trichoderma, Pseudomonas, Bacillus, Rhizobia), and (4) chemical methods (organic or inorganic, metallic or non-metallic, insecticides, fungicides, bactericides using coating to form pellets or entrustments). Methods can also be combined to ensure pathogen inactivation, next to addition of auxiliary materials like nutrients or other growth promoting agents. In the past years, the application of chemical seed dressing has declined due to suspected negative effects on diversity of organisms agricultural landscapes [102, 261]. Growing concerns has led to a banned of most insecticides in the European Union, as well as chemical seed dressing using the agent Thiram (TMTD), widely applied as a fungicide in rape and leguminous seed treatment to prevent soil‐borne infections [52].

Application of alternative non-chemical seed treatment methods are propagated, like electron beam treatment or are under development like cold plasma application in pre-harvest. Most non-chemical alternatives are not functional against soil borne pathogens or insect or animal grub, because no long-lasting reservoir of agents is formed. Commonly found soil borne pathogens, which often can also be spread by invested seeds, include the fungal genus Fusarium, Pythium, Rhizoctonia, Phytophthora, Verticillium, Rhizopus, Thielaviopsis, and Sclerotia [235]. In addition to being soil borne, some pathogenic bacteria like Ralstonia solanacearum, Streptomyces scabies, Clavibacter michiganensis subsp. sepedonicum, Pectobacterium spp., Dickeya spp., and Agrobacterium tumefaciens are also transmitted through infected planting materials such as tubers and cuttings. Some soil borne viruses, such as Tomato mosaic virus (tomato), Tobacco ringspot virus (tobacco), and Indian peanut clump virus can also be transmitted by nematode vectors or via infected seeds [235].

Nevertheless, the major strength of non-chemical methods lies in the prevention of seed borne infection originating from surface, or near surface attached pathogens. Currently, there are 213 annotated seeds borne pathogens according to the ISTA Pest list [11], encompassing fungi, bacteria and viruses. Commercially relevant examples are Fusarium causing a number of diseases in various plants (head blight in barley and panama disease of banana), Tilletia causing common bunt, dwarf bunt and stinking smut of cereals, Ustilago tritici causing common and loose smut in barley and rye, Phoma causing stem rot in rapeseed, Typhula incarnata causing snow mold in rye, and Pyrenophora graminea/Drechslera tritici-repentis, causing yellow leaf spot in wheat and barley.

Apart from the localisation of the pathogen on or inside the seed, the complex lifestyle with sexual and asexual cycles of especially fungi makes seed treatment more complicated. Reproduction of fungi is primarily by means of spores which can be produced sexually or asexually. The sexual reproduction cycle (teleomorphic phase) of fungi forms different types of spores via meiosis such as oospores, zygospores, ascospores and basidiospores. In the asexual cycle (anamorphic phase) oidia (formed by fragmentation of hyphae into individual cells), conidia (borne on tips or sides of specialized branches of hyphae) and sporangiospores (a nonmotile spore born in a sporangim or case) are produced by mitosis [2]. The disease cycle of monocyclic fungi usually starts with a primary infection, which involves colonization, growth, and reproduction as well as overseasoning in the absence of the host. Polycyclic fungi on the other hand, produce asexual spores (secondary inoculum) at each infection site that can cause new (secondary) infections to produce more asexual spores for more infections.

In vegetables and herbs, bacterial and viral pathogens are of special concern. Bacterial wilt in tomato caused by Clavibacter michiganensis, Xanthomonas causing citrus canker, bacterial leaf spot in many plant species, black rot of crucifers and bacterial blight of rice, Pseudomonas syringae causing wilt and spot diseases in many vegetables and legumes are frequently reappearing. Viral pathogens in vegetables and herbs encompass the mosaic virus (TMV, ToMV), Asparagus Virus (AV-2), tobacco ringspot virus (TRSV) and pea early browning virus (PEBV): The efficiency of cold plasma in reducing seed associated pathogens will be discussed in the following Sects. 6.2.2, 6.2.3 and 6.2.4. Not considered in this chapter are losses in seed quality and health caused by nematodes, insects, herbivory, nor post-harvest disease, which will be handled in Sect. 6.4. Notably, almost every study on CAP inactivation of pathogens is unique because they either use a specific plasma source, often build in-house, with specific configurations (e.g., input power, working gas, treatment time), they deal with treatment of different matrices (e.g., suspensions in water, other solutions, seeds from different plant families) and different type of pathogens (fungi of different life cycle stages, bacteria in sporulated or vegetative form, viruses) are used. This diversity makes it difficult to compare results from different studies directly and to define any universal inactivation parameters.

6.2.2 Effect of Cold Plasma Treatment on Fungi

Because of their relevance for losses in crop yield, fungal pathogens have been subjected to a number of studies, 39 are listed in Table 6.1. Inactivation of is highly dependent on the treatment properties, and the optimal parameters need to be chosen on a case-by-case basis.

Table 6.1 Efficiency of non-thermal plasma treatment for inactivation of fungal pathogens in pre-harvest
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Studies, which can serve as a general proof of concept for inactivation of fungal pathogens, are using spore suspensions as a test object. In these cases, the complex matrix of seed surfaces including topography, texture and chemical composition are absent. Moreover, information on the effect of the individual CAP treatment on seed germination is lacking, which makes it difficult to transfer gained knowledge to actual occurring crop diseases. However, 12 studies demonstrated efficient inactivation of Alternaria, Ascochyta Aspergillus, Chaetomium, Cladosporium, Colletotrichum, Fusarium (Gibberella Penicillium,), Phomopsis and Rhizoctonia. Reduction was in the range 44% to complete inactivation [13, 127, 143, 146, 193, 232, 243, 250, 252, 254, 316, 320, 360]. A variety of plasma sources was used including DBD in three cases, jets in two, corona, and arc discharge in three cases, microwave induced, and radiofrequency CAP was applied in two cases each.

Several authors used artificial inoculated seeds to investigate the inactivation efficiency of CAP. Important for pre-harvest application is an unimpaired seed germination, making it necessary to at least monitor maximum germination for the respective plasma treatment. Unfortunately, this was not always the case, but needs to be addressed in future studies. Fungal pathogens investigated belonged to the genus Alternaria, Aspergillus, Cladosporium, Colletotrichum (syn. Glomerella), Didymella (syn. Mycosphaerella), Kabatiella, Penicillium Rhizoctonia, Stemphilium, Trichothecium. Studies with no simultaneous determination of seed published inactivation of pathogens from 1 to 5 log units for Aspergillus [25, 56, 58], by ~3 log units for Penicillium on barley seeds [189], a reduction in Fusarium infected seeds by 20–80% [146], or a significant decreases in Cladosporium diseased plants [193]. No decrease in seed germination was detected in four studies using artificial inoculated pathogens and subsequent CAP treatment. Efficiency of inactivation was depended upon the pathogen used with no effect for Fusarium inoculated on maize seeds [84], 0.5–3 log units for Penicillum and Aspergillus [301], 80% reduction of viable spores of Fusarium on rice seeds [143], up to complete inactivation of Cladosporium on cucumber seeds [325].

On the other hand, significant decrease in seed germination after CAP treatment was reported by seven studies. Efficiency of CAP treatment again depended on the pathogen used, along with plasma source and applied parameter including treatment time. Effects ranged from no effect for Colletotrichum/Glomerella along with rather small reduction in seed germination <10% [81]. A reduction by 3 to 4 log units for Aspergillus, Alternaria and Fusarium on wheat seeds up to complete inactivation using the same plasma source for Fusarium, Trichothecium and Aspergillus on maize seeds which was accompanied by severe decrease in seed germination for longer treatment times in both cases [379, 380]. A delay in seed germination >50% for Chinese cabbage accompanied by nearly complete inactivation of Rhizoctonia [241]. Seed germination of wheat and barley was decreased by ~54% for treatment times >120 s, while at the same time DBD treatment led to a nearly complete reduction of Fusarium [117]. Lentil seed germination decreased by 95% after treatment time of 240 s with a coplanar DBD, at the same time reducing viability of Penicillium and Aspergillus by 3 and 1.6 log units/g seeds, respectively [356]. Additionally, a complete loss of pine seed germination for coplanar DBD treatment >60 s was detected, while a complete inactivation of Fusarium was observed [307].

However, 18 studies dealt with natural fungal communities on seeds (often accompanied by artificial inoculation with specific fungi), which includes also non-pathogenic fungi. Two studies specifically focused on natural occurring pathogens like Diaporthe/Phomopsis complex on seeds of soybean [264] and Fusarium sp., Stemphilium sp., Colletotrichum/Glomerella, Didymella pinode on seeds of narrow-leaved lupine [81]. Pérez Pizá and colleges [264] published reduction in Diaporthe/ Phomopsis infected soybean seeds from 15% to minimum of 4% after DBD treatment with no decrease in seed germination. Moreover, Filatova and colleges [81] reported the efficacy of at 15 min treatment of lupine seeds using a radiofrequency (RF) capacitively coupled discharge with maximal reduction of 16% for Fusarium, 14% for Didymella, 10% for Stemphilium and no reduction of Colletotrichum/Glomerella. At the same time, CAP treatment did not decrease field emergence at 15 min treatment time, while 20 min treatment resulted in a decrease by ~7%. Four publications dealing with natural fungal communities present a detailed identification applying selective plating and visual determination methods or next generation sequencing. The first one by Filatova and colleges [82] identified fungi on lupine and pea seeds using morphological and cultural characteristics. Fungi on lupine consisted mainly of Fusarium and Alternaria, while on seeds of field pea Fusarium, Alternaria and Stemphilium were identified, using cultring techniques, which are selective and don’t include the whole community. Moreover, inactivation using 10 min treatment of a radiofrequency capacitively coupled discharge displayed a maximum reduction of 4%, 24% and 3% for Fusarium, Alternaria and Stemphylium on pea seeds respectively. On lupine seeds, maximum reduction occurred at 15 min treatment time resulting in ~9% and 1% for Fusarium and Alternaria respectively. Seed germination in the laboratory resulted in no decrease until 15 min CAP treatment for field pea and a decrease by 1% at 15 min for lupine seeds. The second study using selective plating identified mainly Aspergillus and Penicillium on seeds of common bean [286]. Treatment applying DBD for 10–30 min revealed complete inactivation of both genera detected. However, seed germination presented as visual radicle formation resulted in a complete loss of radical formation at 20 and 30 min CAP treatment and in an inferior radicle development at 10 min treatment time. Two further studies implemented next generation sequencing to disentangle the fugal community. Lee and colleges [174] focused on ginseng seeds, detecting the following genus Coniochaeta, Pyrenochaeta, Humicola, Clonostachys, Fusarium, Mortierella. Treatment using DBD for 10 min three days in a row showed no reduction in Humicola and Clonostachys, a reduction below 20% in Fusarium and Mortierella and a reduction by >80% in Coniochaeta and Pyrenochaeta in an Argon/oxygen mixture. Additionally, no decrease in seed germination was observed. Likewise, next generation sequencing, as well as plating and visual identification were applied by Mravlje et al. [229] on the fungal community of buckwheat seeds. Alternaria, Didymella (Phoma), Epiccocum, Rhodotorula and Hannaella were identified. A radiofrequency plasma system operated at low pressure of 1 Pa was implemented and treatment times were in the range of seconds. After 120 s treatment, filamentous fungi of the genus Alternaria predominated, while other genus was detected in lower quantities. Alongside, seed germination decreased by ~10% from 15 to 45 s and by 50% at treatment times >45 s. No in-depth identification of the natural fungal load was presented in 12 other studies, displaying inactivation efficacy on barley, broccoli, sweet basil, hazelnut, maize, pea, rapseed, rice, soybean, wheat as a bulk parameter. Inactivation was in the range of 10% to 3 log units (99,99%) inactivation [7, 37, 58, 145, 150, 151, 157, 161, 189, 271, 379, 380]. Differences in the susceptibility of fungi to CAP compared to bacteria were previously reported with fungi being more resistant to CAP exposure [174, 189, 270, 380]. Nevertheless, the proposed mechanism of inactivation of filamentous fungi by CAP likely resemble the ones described in bacteria (see Sect. 6.2.3).

The plasma-treated fungal spores often show severe morphological degeneration including damage of cell envelope structures [252] also related to lipoperoxidation of cell macromolecules [316] and seem to undergo necrotic death [250]. Panngom and colleges [250] argued that elevated levels of peroxynitrite and nitrite originating from the CAP treatment of the saline solution might have been responsible for the observed fungal spore death. Furthermore, when direct CAP treatment is applied inactivation can occur via different other mechanisms e.g. DNA fragmentation or destruction by UV irradiation, erosion through intrinsic photodesorption or erosion through etching to form volatile compounds as a result of slow combustion using oxygen atoms or radicals emanating from the plasma (reviewed by [165, 217, 218, 228]). As noted before, CAP produces different reactive species (RONS, e.g. atomic oxygen (O), metastable oxygen \(\left( {{\text{O}}_2^* } \right)\), superoxide \(\left( \cdot {{\text{O}}_2^- } \right)\), ozone (O3), hydroxyl radical (·OH), hydrogen peroxide (H2O2), nitride oxide (NO), and nitride dioxide (·NO2)), which play a crucial role in the inactivation of any microbial target by oxidation of cytoplasmic membrane, protein and DNA [229]. Most likely, O and ·OH induces the largest number of hydrogen abstraction reactions [55, 364], while the activity of HO2 and H2O2 is lower, however, the number of main chain and branched chain fractures of cell wall glucan structures appears to be bigger. Consequently, the destructive effect of H2O and H2O2 is more efficient [55]. Fungi might exhibit possible protection from CAP damage when carotene pigments are present protecting spores from oxidative damage by plasma [232].

6.2.3 Effect of Plasma Treatment on Bacteria

Inactivation of bacterial pathogens, like fungal ones, is highly dependent on plasma source, configuration, and the treatment properties. The majority of studies applied DBDs for direct treatment or gliding arc for indirect treatment by producing plasma treated water or gas (Table 6.2). There are several proof of concept studies using spore suspension of phytopathogenic bacteria (e.g., Xanthomonas campestris, Erwinia sp., Clavibacter michiganensis, Pectobacterium carotovorum) showing a successful reduction in the number of viable bacteria from 1.5 log units to complete inactivation in a time-dependent manner [223, 224, 227, 230, 344].

Table 6.2 Efficiency of non-thermal plasma treatment for inactivation of bacterial pathogens in pre-harvest
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There is an almost equal part of studies dealing with pathogens artificially inoculated on seeds or growth solution and naturally load on seeds with the majority applying DBD plasma sources or jets. Artificial inoculation of hydroponic growth solution for tomato cultivation with the pathogenic bacteria Ralstonia solanacearum and subsequent treatment of this solution using a gas-liquid phase discharge plasma reactor displayed a reduction by 5 log units in the solution and a decrease in disease severity of tomato seedlings by 80% after 10 days of growth [247]. Treating tomato seeds with a capacitively coupled plasma (CCP) generated by a radiofrequency discharge at 150 Pa led to an increased resistance of the 30 days old plants to Ralstonia solanacearum by 25% [140]. Treating seeds which were artificial inoculating with either non-plant-pathogenic bacteria Bacillus atrophaeus and Escherichia coli as a model or with actual pathogenic bacteria, e.g. Xanthomonas, Burkholderia plantarii and Geobacillus stearothermophilus often resulted in an efficient reduction of viable bacteria from 2.4 to 6 log units, but simultaneously reduced seed germination in one case [189]. For two studied no information on seed germination after plasma treatment was presented for the same study [42, 242]. Altogether, vegetative cells of Bacillus atrophaeus and Escherichia coli seemed to be easier to inactivate than spores of Bacillus atrophaeus [189]. Disease severity was monitored in one study using artificially inoculated Burkholderia plantarii on seeds of rice which were subjected to atmospheric pressure plasma jet, subsequently [246]. Results indicated a reduction in disease severity of seedling blight by 40% along with no reduction in seed germination.

Six studies presented information on the effect of CAP on natural bacterial load on seeds, which of course also encompasses non-pathogenic bacteria. The natural community was identified in two of the six studies [174, 270]. A very recent study by Lee and colleges [174] applied next generation sequencing to elucidate the community on seeds of ginseng identifying the genus Kocuria, Variovorax, Pseudomonas, Duganella, Rahnella, Flavobacterium, Azospirillum and Chryseobacterium. CAP treatment for thre times 10 min using a DBD releveled a reduction by less than 20% for Pseudomonas and Duganella, as well as a reduction by 30–65% for all other, while a t the same time no negative effects on seed germination were detected. Puligundla and colleges [270] actually focuses on post-harvest relevant bacteria and therefore used general and selective growth media to quantify B. cereus, E. coli, Salmonella spp. on rapeseed. For all detected microorganisms the reduction after treatment with a corona discharge plasma jet for 3 min. was in the range of 1.2–2.2 log CFU/g. However, 3 min CAP treatment also provoked a decrease in seed germination by ~30%. The four remaining studies dealt with an unknown community of natural bacterial load on seed surfaces of sweet basil, barley and wheat as well as chickpea and maize showing inactivation by 1.2–3 log units, up to complete inactivation [380] or a reduction by 30% in contaminated seeds [7, 189, 216]. In two of the latter studies, seed germination was severely negatively affected with a decrease by up to 60% [189, 216].

Altogether, seed decontamination/ inactivation often was accompanied by a reduction in seed germination when applying identical CAP treatment times, impeding a possible application of CAP in pre-harvest seed treatment. It has to kept in mind, that plasma can induce a sub-lethal state of bacteria leaving them viable but nonculturable after CAP treatment (VBNC) state [367]. Further investigations on this effect are needed. Previous studies investigating the effect of CAP treatment on bacteria as well as fungi on seed surfaces demonstrated that bacteria especially in vegetative state are more prone to CAP exposure compared to fungi[174, 189, 270, 380].

Some authors investigated and proposed inactivation mechanisms, which resemble some of those found for fungal inactivation. Previous knowledge originating form plasma medicine and/or food science can be transferred regarding some general patterns and concepts for inactivation. Different effects of CAP treatment were observed for Gram-positive and Gram-negative bacteria [147, 167, 198] that differ in cell envelope structures. Gram-negative bacteria, which possess a cell wall composed of an outer membrane and thin peptidoglycan (murein), displayed substantial damage to the membrane resulting in the cytoplasm leakage. Gram-positive bacteria on the other hand, display cells with a thick cell wall and did not show the significant morphological modifications and decontamination was most probably appeared here due to interactions of reactive compounds with the intracellular components [147, 238]. Bacteria morphometry might also be responsible for differences in inactivation patterns with more resistant spherical cells (cocci) than rod-shaped cells (bacilli) [167, 331]. As pointed out before, CAP produces many reactive oxygen and nitrogen species (RONS), which can oxidize proteins, lipids, and nucleic acids and lead to pathogen destruction [180]. Moreover, inactivation mechanisms might include erosion the surface of microbial cells through etching [218], oxidative damage of intracellular macromolecules, such as membrane lipids, proteins, and DNA, and a reduction in intracellular pH from diffusion into the microbial cells disrupting pH homeostasis [166]. Furthermore, sub-lethal damages can induce viable but non-culturable (VBNC) states in fungi as well as bacteria which is defined as an inactive form of life that is induced by stressful conditions [51] and undergoes recovery under suitable conditions [277]. These state transitions have been reported after CAP treatment [53, 72, 197, 298] and need to be taken into account in future studies.

6.2.4 Effect of Plasma Treatment on Viruses

Plant virology is a very dynamic research area with new plant viruses being detected more rapidly. Moreover, awareness of their pathological impact and severity of economic loss caused by reduction in yield by up to 100% [222, 312] or quality of crops has led to efforts for new detection as well as plant treatment methods. Plant pathogenic viruses are mainly transmitted horizontally by biological vectors, usually insects, but can also be transmitted via seeds, tubers, rhizomes and bulbs [294]. Increasing evidence suggest that transmission can also occur via contaminated process water [205].

The majority of studies dealing with the effect of CAP on plant viruses applied DBDs in various configurations, next to jets and torches for direct treatment and indirect underwater treatment (Table 6.3) of viruses in suspension and inoculated onto plant leaves. Only one study dealt with actual seeds, cucumber and pepper, which were naturally infected with cucumber mosaic virus, zucchini yellow mosaic virus and watermelon mosaic virus [325]. Štěpánová and collegues used only one treatment time per plant species (20 s for cucmber and 4 s for pepper) and detected no decrease in viral load after treatment of seeds with a diffuse coplanar surface barrier discharge plasma (DCSBD). Seed germination on the other hand, was not decreased after plasma treatment. Milusheva and colleagues [212] investigated the effect of a surface-wave-sustained argon plasma torch and an underwater diaphragm discharge on plum tree microplants, which were naturally co-infected by M and D strains of Plum pox virus (PPV). Microplant’s nodal segments or leaflets were subjected directly to a CAP torch, as well as to electrical discharges in water media. Treating nodal segments without leaves in gas medium using the torch tip tuned out to be most effective with no detection of viable D strains of Plum pox virus along with a decrease in symptomatic plants by 80%. Plant leaves inoculated with specific viruses were the focus of two studies using Tulane virus for Romain lettuce and tobacco mosaic virus for tobacco [104, 213]. Reduction of Tulane viral load by 1.3 ± 0.2 log PFU/g Romanian lettuce and no necrotic lesions cause by tobacco mosaic virus detectable in plant leaves after treatment. Filipić and colleges [86] investigated irrigation water inoculated with Potato virus Y wich was treated using a single electrode cold atmospheric plasma jet. No infection was detected in the plant infectivity assay using Nicotiana tabacum cv. ‘White Burley’ from 15 min treatment time of suspension onwards.

Table 6.3 Efficiency of non-thermal plasma treatment for inactivation of viral pathogens in pre-harvest
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Possible mechanisms for viral inactivation were presented in a study by Guo and colleges [100], who did not investigate plant viruses but bacteriophages T4, Φ174 and MS2, which can serve as a general proof of concept for inactivation. Bacteriophage suspension was treated with PTW produced by an air surface microdischarge, showing an inactivation below the detection limit after 120 s treatment. Although not being pathogenic to plants, the proposed model of inactivation is likely to be adaptable from bacteriophages. The proposed model of inactivation includes plasma-generated reactive species, especially singlet oxygen, which efficiently inactivated different kinds of bacteriophages in water, including double-stranded DNA, single-stranded DNA, and RNA bacteriophages by damaging both nucleic acid and proteins and leading to excessive aggregation of the bacteriophages. In addition, knowledge can be transferred form studies dealing with other types of viruses, e.g., animal viruses. Work on Newcastle disease (ND), an infectious viral disease of avian species, reported complete inactivation after PTW treatment resulting most likely from singlet oxygen, which quickly reacts with cysteine, resulting in the formation of cystine (R-cys-S-S-cys-R) with disulfides; thus creating products which lead to aggregation of bacteriophages [362]. Furthermore, enzyme activity can be impaired by hydroperoxides which is formed by the interaction of amino acids, including tyrosine, tryptophan, and histidine, which selectively interact with singlet oxygen [63].

Altogether, studies dealing with the efficiency of CAP to inactivate plant pathogenic viruses on seeds and plants are scarce and efforts should be taken to fill the gaps of knowledge. Unknown up to now is the effect of CAP itself on insects as transmission vectors, which should be examined in the future. In addition, in natural environments mixed-infections with two or more plant viruses are frequent, with viruses being able to interact in multiple and intricate ways. These interactions can be synergistic, antagonistic, or neutral and will likely have an impact on the efficiency of CAP application for phytosanitary purposes.

6.3 Application of Non-thermal Atmospheric Pressure Plasma to Seed Germination and Plant Growth

Major seed dressing methods are aiming to prevent pathogenic attack and outbreak by using e.g. fungicides. Inoculation of seeds with fertilizers, chemical stimulants or plant growth promoting bacteria (PGPB) support seed germination performance to promote proper seedling establishment, further plant growth and stress resilience to finally secure or increase yield. Furthermore, different kinds of chemical and physical seed treatment methods have been studied aiming to stimulate and synchronize germination of seed population and to prime plants against various stresses [9, 78, 251]. Numerous studies have shown that plasma as a physical treatment method can improve seed germination performance and plant growth (Tables 6.4 and 6.5). Recent studies investigated the potential of plasma to prime seeds against biotic and abiotic stressors as well [14, 17, 80, 84, 99, 178, 219, 264].

Table 6.4 Effects of atmospheric pressure plasma on plant seeds
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Table 6.5 Effects of low-pressure plasma on plant seeds
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Unlike in plasma medicine, there is a much greater variability of plasma sources and a higher number of plant species to be treated. In contrast to human or animal tissue surfaces, the surface of seeds consists of dead cellular material and water-repellent polymer layers to protect the plant embryo from physical and chemical influences [26]. Another difference is that, in contrast to animal organs, the entire seed is treated, not single specific parts of it. In addition, seeds are not treated as a single individual, but usually in a batch with a large number of seeds at the same time. Therefore, there is a need to develop devices for treatments on a larger scale, which will be necessary for future agricultural application. Thus, the requirements for plasma source dimensions to treat plant seeds along with a greater flexibility of plasma processes and operation conditions need to be addressed. Section 6.3 focuses on gaseous plasma treatment of seeds under atmospheric and low-pressure conditions comprising the plasma effects on physicochemical alterations of the seed and on germination and developmental processes.

A wide range of options exists to generate non-thermal plasma. This refers to configuration of electrodes, applied pressure, feed gas composition and flow rates, and electrical parameters (voltage, type of electrical current, frequency, power) used to ignite plasma, as well as treatment times and the mode of treatment with respect to direct or indirect plasma exposure of the plant target, as can be seen in Tables 6.4 and 6.5. In general, dielectric barrier discharges (DBD) in different configurations such as surface DBD (planar DBD) or diffuse coaxial DBD (DCSBD), gliding arc discharges, jets, corona discharges, microwave discharges as well as different kinds of radio-frequency (RF) discharges exist and has been applied. For treatment of seeds under atmospheric pressure, dielectric discharges using AC, DC or even RF were most frequently studied so far (Table 6.4). Regarding low-pressure conditions, RF plasmas were mostly investigated (Table 6.5).

Proper seed germination and seedling establishment on the field is the fundamental requirement for resilient plant growth, which ultimately determines the yield. Here, plasma has relevance for potential future application in agriculture as many studies have proven the beneficial effects of non-thermal plasma on seed germination performance. Important agricultural relevant plant species with different usages ranging from food and feed production to pharmaceutical and plant-based industry have been investigated so far (Tables 6.4 and 6.5). Wheat (e.g. [38, 99, 207]), maize (e.g. [381]), rice (e.g. [150, 373]) and barley (e.g. [38, 267]) produce seed-like fruits (botanical term “caryopsis”; caryopses are propagation units and the term “seeds” will be used within this chapter for simplification) containing a starchy endosperm important for feed and food production. Legume seeds such as soybean (e.g. [175]), pea (e.g. [151, 330]), chickpea (e.g. [216]), common and mung bean (e.g. [35, 281, 287]), and lentil (e.g. [34]) belong to staple food, while alfalfa [80], blue lupine or clover (e.g. [211]) are used for feed production and are relevant in crop rotation because of the symbiotic activity with nitrogen-fixing bacteria. Increase in seed germination after plasma treatment has been also detected for seeds from rapeseed (e.g. [178, 270]) or sunflower (e.g. [203, 371]) known as oil plants. Moreover, this also applies for seeds from vegetables (e.g. radish [202]); spinach [136, 310]; tomato [196], zucchini [151], spices (e.g. pepper, [325, 341]), herbs (e.g. coriander, [135]; sweet basil [7, 317]), pharmaceutical relevant plants (e.g. ginseng, [174]); ajwain, [92]; hemp, [306]; safflower, [69] or trees (e.g. empress tree, [269, 389]); Norway spruce [258] and black pine [309].

6.3.1 Plasma Effects on Seed Surface Morphology

Depending on the plasma intensity of direct plasma treatment mode, outer seed surfaces can be modified leading to cracks, holes and fissures caused by etching and erosion events. Optical analysis by Scanning Electron Microscopy (SEM) is frequently applied to detect changes on seed surfaces by atmospheric DBD or low-pressure RF plasma treatment.

Several studies using DBDs documented surface modifications of wheat seeds after non-thermal plasma treatment [99] detected cracks on seed surface after 4 min air plasma treatment. [207] showed etching effects on the seed coat, which occurred after the air, nitrogen and argon plasma treatments, causing the change in hygroscopicity and permeability of the wheat seed. Li et al. [179] observed gradual destruction of square mesh structures and occurrence of cracks with elevated treatment time of air plasma. Molina et al. [220] found that the seed pericarp was progressively etched and damaged with increasing helium plasma exposure. Changes started with random nano‐grooves on the outer layer at treatment times of 5 min, which extended when the treatment time was further increased to 15 min.

Moreover, other plant species were subjected to DBD treatment like barley, pea, thale cress or quinoa [256] investigated barley seeds and reported that plasma treated seed surface were etched and eroded after nitrogen/air plasma treatment for 40 s. Pea seeds were used by Gao et al. [89] displaying distorted and partially destroyed surfaces and ridges on the seed epidermis which gradually dissolved caused by seed coat erosion via bombardment of seeds with free radicals and ions of air plasma treatment at 15 W for 3 min. In addition [330], applied air DBD treatment to pea seeds and observed an uneven disruption, abrasion or even loosening of original structures in testal areas near the plumule- and radicle apex, especially after 10 min exposure. Effects of plasma on the model plant thale cress has been studied as well [54] presented dose-dependent etching effects of air plasma on seed surface encompassing slight shrinkages at 1 min treatment time up to detached epidermis at 10 min plasma exposure. Similarly, Bafoil et al. [16, 18] found changes on the seed surface after air plasma treatment for 15 min. The authors observed a physicochemical etching of the surface by plasma treatment due to rearrangement of macromolecular structures and exudation of lipid compounds from the seed.

In further studies, no damage (e.g. cracks, holes) of seed surface structure was observed after plasma treatment using a DBD plasma sources, e.g. pea [334], radish [159], spinach [136], sweet basil [7], maize [380], onion [340] or wheat [190].

Low-pressure plasma is also able to modify seed structure (Table 6.5). Flax seeds experienced etching of the cuticle and an accompanied weakening of the underlying mucilage secretory cell (MSC) walls [61]. Although longer RF plasma treatments (15–20 min) induced extensive cracking of the outer integument, the water uptake was not affected [281]. reported a rougher seed surface and an increasing amount of material being removed at 20 Pa with elevated treatment times, which resulted from energetic ions that impinge on the surface. On safflower seeds treated with low-pressure argon RF plasma for 130 min, changes in seed structure and a smoothening relative to the untreated control seeds appeared [69]. Quinoa seeds displayed plasma etching affecting the pericarp after non-thermal plasma treatment [96].

In general, observed modifications of seed surfaces after plasma treatment can be related to following factors: particle bombardment of highly energized species, local heat generation and possibly the individual nature with respect to heterogeneous morphological structures and chemical compositions of outer seed layers. Overall, there are critical methodological aspects for the visual detection of these changes by SEM: 1. SEM analysis provides only a spatially limited section of the entire seed surface. 2. Most seed surfaces are not homogeneous but are highly structured, requiring extensive surface analysis. 3. Eventually, a great number of single SEM pictures have to be analyzed, in addition to different individual seeds to conclude generalizations. 4. SEM analysis of seeds is performed in high or low vacuum and hence plasma effects on seed surfaces could be intensified.

6.3.2 Chemical Modification of the Seed Surface

During direct plasma treatment, bombarded by exited particles such as radicals and ions that can lead to erosion, etching and even chemical modification of the seed surface. This changes the chemical structure and morphology of the surface (e.g., roughness). Interaction of electrons and ions with outer surface layers result in modification and finally higher wettability [34]. Chemical modifications provoked by treatments using gaseous plasma that contain certain proportion of oxygen have been detected in several studies in which oxidation of seed surfaces irrespective of plant origin was observed (e.g. wheat, barley quinoa; [38, 96, 219, 220, 240, 325]) (see also Tables 6.4 and 6.5). Three different methods have been applied to study potential chemical modification: (1) X-ray photoemission spectroscopy (XPS), (2) attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy (ATR-FTIR) and (3) Time of Flight-Secondary Ion Mass Spectrometry (ToF-SIMS). Mainly XPS has been applied to detect chemical changes, because this method can detect elements and their chemical state (e.g. oxidation and binding energy) located at seed surfaces, such as carbon, nitrogen, oxygen, potassium, sodium, magnesium and calcium (e.g. [96, 99, 116, 281, 325]). A correlation between relative oxygen abundance and plasma exposure times from 10 s up to 15 min were noticed on wheat seed surfaces [220]. The increased carbon-oxygen bonds were attributed to air impurities of the DBD device operated using helium at 5 slm. [380] analyzed maize seed surfaces using ATR-FTIR after exposure to DBD treatment for 60 s and found an increased occurrence of polar nitrogen and oxygen containing groups. RONS generated by DBD led to oxidation of lipids located at pea seed surface and subsequent increase in water uptake performance of peas [334]. The decrease of C-H bonds, typical for fatty acids, was more pronounced when air or oxygen was used as feeding gas. However, it was not stated if band intensity typical for oxygen containing groups were increased in FTIR spectra. Bormashenko et al. [34] analyzed the surfaces of wheat and lentil seeds treated with low-pressure air RF plasma using ToF-SIMS and found that a higher proportion of O-containing groups and N-containing groups could be detected on seed surfaces.

6.3.3 Alterations of Seed Surface Hydrophobicity

Physicochemical alterations of seed surfaces can result in changes of surface hydrophobicity. Wettability of seed surfaces can be estimated by measuring the water contact angle (WCA) of a tiny water droplet placed onto the surface. Depending on the contact angle, surfaces are referred as hydrophilic (<90°) or hydrophobic (>90°) [87, 168]. The WCA of plant seed surfaces are usually above 90° but can range from 130° to 76° depending on plant species [315]. Increased wettability of seed surfaces are observed after direct plasma exposure in e.g. wheat [34, 71, 190], soybean [175, 264], rapeseed [178], maize [380], lentil [34, 357], bean [34, 281] or barley [38]. Atmospheric pressure plasmas (e.g. DBD, plasma jets) and RF low-pressure plasmas either operating with air [18, 35, 59, 92, 371], oxygen[234, 281], (Piza et al. 2018), nitrogen[115, 119] or with nobles gases argon [38, 49] or helium [5, 175, 177, 219] displayed effects on seed surface wettability. By using a DCSBD system, different applied feed gases (air, oxygen and nitrogen) resulted in similar strong decreases of WCA values of maize seed surface with increasing plasma treatment times from 30 s to 5 min [115]. Comparable changes in surface wettability of wheat seeds by plasma have been reported for various DBD systems working with feed gases air [71, 190, 347], argon [38, 240] or helium [220].

Indirect plasma treatment does not lead to any significant changes in wettability of seed surfaces from wheat [190], Thuringian mallow [259] and rapeseed, barley or lupine [355].

6.3.4 Alterations of Seed Water Absorbance

The seed coat consists of several layers of dead cells. Seeds from several kinds of plant species such as legumes contain a cuticle as the outer layer that is enriched with phenolic compounds and fatty acid derivates resulting in a hydrophobic seed surface. Naturally occurring cracks on seed surfaces of soybean (Glycine max (L.) Merr.) can contribute to water uptake during imbibition [194]). Surfaces of caryopses from wheat or barley contain carbohydrate polymers (e.g., cellulose, hemicelluse) and lignin, which renders the surfaces to hydrophobic state. The mandatory initial step for germination is the uptake of water (imbibition process) to enable physiological processes [244]. Uptake of water does not occur evenly along the seed surface area. In seed science, several methods exist to deduce the route of water entry to the inner parts of the seeds, e.g., using dyes or stable isotopes as tracer. Furthermore, prior to imbibition tests, seed structures can be blocked by water impermeable material or dyes and thus can be used to trace the influx of water [65, 186, 385]. Bafoil et al. [17] measured seed permeability of thale cress by absorbance of tetrazolium red. This test is based on the enzymatic oxidization of tetrazolium red by dehydrogenases in the respiratory chain. Interestingly, the permeability was decreased by plasma treatment which seems to be contradictory to most of published research upon seeds.

Soybean seeds exposed to different gaseous plasma such as DBD displayed increased water absorption after one hour of imbibition [335]. The observed alterations were correlated with treatment time from 30 to 120 s and were more pronounced for nitrogen containing plasma compared to air and oxygen plasma. Similar observation using the same experimental setup was found for one hour imbibed pea seeds treated for 60, 180 and 300 s [334]. Wheat seeds with higher water uptake after plasma treatment simultaneously displayed a decrease in weight due to plasma etching process proved by SEM analysis [220]. Interestingly, water uptake of spinach or wheat seeds was unaffected after plasma treatment even though strong decrease in seed surface hydrophobicity was observed [138, 190].

Future research on plasma treatment of seeds should consider analysis of seeds by nuclear magnetic resonance (NMR) or magnetic resonance imaging (MRI). These techniques give more detailed information about water imbibition process as distribution of water under real time conditions can be monitored [33, 128, 263]. These techniques could provide a better understanding of the plasma-induced effects on whole-seed water permeability.

6.3.5 Plasma Effects on Seed Germination and Plant Growth Parameters

It is assumed that enhanced wettability due to physicochemical seed surface modifications is one of the major factors improving seed germination performance. The stimulating effects on seed germination is frequently discussed with the ability of plasma to break physical dormancy by reducing seed coat hardness and increased water permeability (e.g., [6, 54, 59, 304]). Plasma treated seeds from e.g. artichoke [119], cumin [280], lentil [34], mimosa [59], water melon [191], mung bean [287], pea [330], rapeseed [176], rice [45, 287], wheat [285, 379], or quinoa [96] had a higher water uptake accompanied with faster germination (see also Tables 6.4 and 6.5).

Analysis of germination kinetics to evaluate the effects of plasma on germination performance essentially involves visual monitoring of germination during different time points. The counting of germinated seeds is based on the macroscopic visible emergence of the radicle protruding from the seed [28, 163]. In studies, 1–2 mm minimal radicle length of germinating wheat and barley, [38, 189], half of the length of germinating soybean [175] or approximately 5 mm radicle length of germinating rice and sunflower [150, 322] were defined for germination. The germination value of a defined time point is presented as germination percentage or (cumulative germination percentage) and often referred as ‘germination rate’ or ‘germination potential’ in literature. Germination potential and germination rate of control and RF plasma treated wheat seeds were determined at day 3 and at day 7 of germination, respectively [139]. The values for germination rate were slightly higher compared to the germination potential values. Here, 80 W plasma treatment influenced both germination values significantly positive in comparison to unaffected 60 and 100 W plasma treatment. On the other hand, soybean germination potential after 3 days and germination rate after 7 days of control and plasma treated seeds did not vary from each other [175]. This indicate that final germination was almost reached after 3 days of germination and plasma treatment had no effect on the final germination of soybean. Treatment of black gram seeds for 120 s with air DBD plasma under low-pressure improved germination rate recorded at day 3 of up to 10% [31]. Moreover, observation time points and intervals vary among studies. The intervals of observation times can range from hours to several days and observation can last up to 3 weeks or longer. The final germination value and the speed to reach maximum germination depends on several plant related factors such as dormancy state and age of seeds along with plant origin and studied cultivar or variety. Soybean [175] or wheat [38] displayed maximum germination within 3 days with ≥80%, irrespective of plasma treatment. In other studies, variation in germination times and maximum germination values can be observed for the same plant species [192, 335], which can be attributed to different applied varieties, cultivars and/or to germination conditions. Molina et al. [220] analyzed wheat germination after plasma treatment using different water supply with 3, 6, and 12 ml. Interestingly, even the longest exposure time of 15 min did not impair seed germination, and seeds from all plasma treatment times displayed the similar maximum germination close to 100% compared to controls after three days. However, plasma treatment times below 2 min resulted in higher germination percentage after 20 and 24 h with 6 and 12 ml water supply. Maximum germination can be affected positively by plasma as shown for e.g., wheat, mimosa or mulungo (Tables 6.4 and 6.5). da Silva et al. [59] found a remarkable increase of final germination for mimosa from 6% for untreated seeds and 50% after 3 min air DBD treatment. Helium DBD treated mulungu seeds had 5% higher maximum germination after 25 days [6]. An increase of more than 10% in final germination recorded after 10 days was observed in wheat after treatment with air DBD plasma for 20 and 30 s [379]. The maximum germination of hemp seeds treated for 5 min with RF air plasma under low pressure was 20% higher compared to untreated seeds [131].

Time-resolved observation of seed germination include several observation times until final germination value is reached, allows more detailed assumptions about velocity and homogeneity of germination. The kinetics of germination can be described with a sigmoidal or logistic function since the rate of germination is not homogeneous over time. The Richard function [105, 283] has been applied to describe plasma effects on maximum germination, median germination time, uniformity and synchrony of germination in hemp [131], lamb's quarter [304], mimosa [59], mulungu [6], rapeseed [176], red clover [130], soybean [175], sunflower [209] and wheat [276, 285].

Next to monitoring of seed germination via observation at several distinct time points, biomass production such as root and shoot fresh and dry weight, lengths of shoot and roots or total seedling lengths are frequently recorded to deduce the effects of plasma. Furthermore, from those parameters different indices can be calculated such as seedling vigour index, and seedling length index (e.g., [309, 330, 335, 379]).

Seedling growth was monitored for thale cress [160], radish [159, 291, 293], sunflower [336, 390], wheat [207], and sweet basil [7]. Soybean seeds were treated with ceramic DBD fed with argon using different voltages and incubation times, and optimum germination was observed in the treatment with 22.1 kV for 12 s [382]. Germination was also higher for up to 1 min treatment and decreased when seeds were treated longer than 2 min [382]. Biomass parameters (shoot and root weight and length) were positively affected in the treatments from 12 s to 1 min and decreased when seeds were treated longer for 2 min [382]. Besides observable positive effects, extensive exposure of seeds to plasma can lead to inhibitory effects on germination and seedling development (Tables 6.4 and 6.5, e.g., [136, 188, 306, 373]). These can be attributed to high levels of radicals and reactive species within plasma such as ozone or nitric oxides (NOx), next to heat and/or high electrical fields leading to deep entrance of electrons to the inner parts of the seeds.

6.3.6 Plasma Effects on Seed and Plant Physiology

Despite the fact that plasma treatment can accelerate germination speed, the simplest explanation for the frequently observed enhancement in seedling growth would be that plasma treated seeds exhibit a time advantage and therefore, higher biomasses of seedling shoots and roots is achieved. However, this would result in similar level of shoot and root growth compared to untreated plants, and thus, shoot/root ratios (or root/shoot ratios) would not be altered. A clear shift of growth to either shoot or root could be monitored for e.g. tomato seedlings [196] and wheat [305, 347]. Moreover, view studies noted alterations of root morphology [123, 142, 178, 196, 280]. The observed alterations on seedling development can be attributed to further effects of plasma components which are different from only physicochemical modification of the seed surface with accompanied wettability and improved imbibition. Here, reactive oxygen and/or nitrogen species (RONS) derived from plasma are the most versatile candidates that can trigger physiological modification and thus have impact on seed physiology with related development and growth processes as well as stress responses [124, 126].

Reactive oxygen species are known to play pivotal role during plant life cycle and are involved in many responses to biotic and abiotic stress factors [121]. During several steps of germination process reactive oxygen species are formed and play a positive role for dormancy release [244]. Externally applied hydrogen peroxide can stimulate pea seed germination with different effects on phytohormone levels of ABA, auxin, SA, JA and cytokinins [24]. In general, primary metabolism, growth and development related as well as stress relevant factors are frequently analysed in plasma studies.

Soybean seedlings six days after seed treatment with plasma showed an increase in levels of soluble protein, ATP, superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and adenosine triphosphate (ATP) and a decrease in malondialdehyde (MDA) [382]. Alterations in antioxidant activities in seedlings of plasma treated seeds were found for various plant species and different plasma exposures as well (see Tables 6.4 and 6.5). Expression of chloroplast ATP synthase subunits was accelerated, and methylation level in ATP a1, ATP b1, TOR, GRF 5, and GRF 6 genes decreased [382]. Altogether, the argon plasma used promoted germination and growth by increasing the concentrations of soluble protein and antioxidant enzymes and regulating the demethylation levels of ATP, TOR, and GRF.

Any observed changes in germinating seeds after plasma treatment is likely a result of the time advantage of germination process, which may be associated with a change in levels of phytohormones. Phytohormones such as abscisic acid (ABA) and gibberellins (GA) are involved in regulation of germination initiation and germination process. ABA plays a role in seed dormancy (and stress responses), and gibberellin contributes to the initiation of germination. Two phytohormones, auxin and cytokinin, play a pivotal role during entire life cycle of plants, and ratio(s) of phytohormones is important for seed germination and seedling development. Moreover, these phytohormones are mandatory for development of shoots and roots.

Total cytokinin content increases during the first two days after imbibition in germinating Tagetes minuta L. seeds and declined during further seedling growth [326, 327]. Similarly, pea, maize, oat, and alfalfa display species specific dynamics of cytokinin levels during germination process and seedling growth [133, 328]. Comparable to phytohormone changes, levels of amino acids and sugars display different pattern during seedling development and growth [44, 79]. Plasma treated dry seeds showed significant changes in phytohormones content and ratios of gibberellins and ABA as well as auxins and cytokinins [1, 99, 175, 179, 209, 256, 330].

Polyphenols are secondary metabolites and belong to markers for different kinds of stresses such as excessive light, heat, drought, flooding etc. Phenolic contents in upper parts of the plants were assessed in barley [319], shoot and roots of wheat [305], spinach [136], and pea [40] after plasma treatment. In wheat, levels of some phenols were slightly increased while others were unchanged or even decreased. In barley shoots, 6 min plasma treatment led to a significant increase in level of total phenolic compounds [319]. When barley seeds were indirectly treated by DBD driven air plasma for 6 min, increase in seedling weight and shoot length as well as increased levels in primary and secondary metabolites, like phenols, in leaves were observed [319].

Analysis of plasma effects on seed germination and seedling growth are mostly undertaken under laboratory conditions. However, proof of concept of stimulating effects of plasma on plant performance needs to be evaluated under agricultural relevant cultivation conditions. These include growth in soil and soil-like substrates but also cultivation in greenhouse and on fields are mandatory. Few studies exist so far performing green house or field trials. Field trials were performed with peanut and rape by Li et al. [176, 177], hemp [130], red clover [211], maize and wheat [84], wheat [123, 139] and maize and pepper [381]. Important traits to evaluate the efficiency of plasma treatment are biomass parameters that are correlated to yield which include number of flowers, number of seeds per plant, seed weight and weight of seeds per harvest area.

6.4 Application of Non-thermal Plasma to Food Sanitation

Food sanitation is the most actively explored area in the application of non-thermal plasma in the food industry. The antimicrobial activity of plasma in vitro has been demonstrated in numerous studies using food poisoning and spoiling microorganisms in planktonic and biofilm states [290]. Furthermore, experimental data are accumulating on sanitation and inactivation of microorganisms contaminating fresh produce, packaged foods, and processed foods, by plasma [346].

6.4.1 Vegetables and Fruits

Post-harvest fruits and vegetables are most frequently examined for microbiological sanitation using non-thermal plasma. Microbial contamination of fruits and vegetables can originate from pre-harvest infection or contamination during storage. To improve the shelf life and storage period of harvested fruits and vegetables, it is essential to inactivate microorganisms. Non-thermal plasma can efficiently deactivate the inoculated microbes and natural microflora on post-harvest fruits and vegetables, as demonstrated in previous studies (Table 6.6). Therefore, it is considered a potential tool for post-harvest sanitation. In most studies, plasma has been applied to fruits and vegetables after artificial inoculation with microorganisms (Table 6.6). However, there are also studies showing the plasma-mediated deactivation of natural microflora associated with fruits and vegetables [32, 93, 107, 164, 183, 221, 272, 338, 363, 366]. Regardless of whether they were inoculated or naturally contaminated with microorganisms, fruits and vegetables were directly exposed to plasma flame or plasma-generated gas. In relatively few studies, plasma-treated water has used for microbial decontamination [363, 366]. Treatment with dry plasma compared to plasma-treated water may be helpful in preventing the introduction of moisture, which can promote microbial growth. Microbial inactivation by dry plasma or plasma-treated water in post-harvest fruits and vegetables shows a proportional increase in response to the treatment time. Roughly about 0.3–7 log CFU reduction depending on treatment time, plasma sources, and feeding gas was observed in most studies (Table 6.6). The difference in inactivation efficiency between bacteria and fungi was not obvious.

Table 6.6 Application of non-thermal plasma to foods
Full size table

Various non-thermal plasma sources such as plasma jets, DBD plasma, gliding arc plasma, corona discharge plasma, and microwave plasma are used for decontamination (Table 6.6). In most of the studies, plasma was generated mostly under atmospheric pressure. However, a group used plasma generated under low pressure [302]. Fruits and vegetables used to analyze the antimicrobial activity of non-thermal plasma are categorized into three groups: fresh fruits such as grape, banana, lemon, strawberry, blueberry, palm, melon, citrus, cantaloupe, and apple; dry nuts such as almond, hazelnut, and pistachio; and fresh vegetables such as corn salad leaves, lettuce, tomato, carrot, black pepper, red chicory, spinach, perilla, mung bean sprout, and argula leaves (Table 6.6). Additionally, Xu et al. [366] investigated the antimicrobial effects of plasma-treated water on button mushrooms and demonstrated that mushrooms had less microorganisms and could be stored for longer after soaking in the plasma-treated water.

Mycotoxin, a secondary metabolite produced by some fungi, is a food contaminant that threatens human and animal health [201]. Fruits and vegetables infected with mycotoxin-producing fungi have recently become a major concern in food safety [329]. Non-thermal plasma is also used to inactivate toxin-producing fungi and remove mycotoxins. The removal and degradation of mycotoxins by plasma in vitro have already been demonstrated in several studies [113, 253, 339]. Studies have also demonstrated that mycotoxins associated with dry nuts and grains, particularly aflatoxin B1, are efficiently degraded by non-thermal plasma[68, 114, 273, 303]. Additionally, mycotoxin levels have been controlled by inactivating producer fungi on fruits and vegetables using plasma. [248] observed that germination of spores and the levels of aflatoxin B2 and ochratoxin A decreased after date palm fruits inoculated with Aspergillus niger were exposed to a plasma jet.

Impact of plasma treatment on the quality of fruits and vegetables as food was analyzed together with antimicrobial activity in most studies (Table 6.6). This analysis is very important to determine whether plasma doses sufficient to kill microorganisms negatively affect the quality of fruits and vegetables as food. The most frequently analyzed quality factors are color, flavor, pH, and antioxidant activity. Studies have demonstrated that maximal antimicrobial efficiency of plasma does not always result in no damage to the food quality of fruits and vegetables. This indicates that there is an optimal plasma treatment condition (mostly treatment time) that produces efficient antimicrobial activity without significant damage to the quality of fruits and vegetables. It may be necessary to identify a proper point balancing between antimicrobial activity and food quality control, even though complete microbial decontamination cannot be achieved.

6.4.2 Meats, Meat Products, and Fishes

Despite their high nutritional value as a protein source, meat, meat products, and fish can be easily contaminated with microorganisms, causing food poisoning and other foodborne diseases [62]. Non-thermal plasma has been used for the decontamination of meat and meat products such as chicken, pork, beef, ham, bacon, and eggs (Table 6.6). Recently, seafood such as fish filets, oysters, and salmon sashimi have been actively explored in plasma applications [47, 122, 255]. In many studies, the antimicrobial activity of plasma was assessed on microbes inoculated onto meats and fish. Several studies have focused on the contamination of natural microflora [88, 275, 345]. Among microorganisms, food poisoning bacteria such as E. coli, L. monocytogenes, Salmonella spp., and Campylobacter jejuni were more frequently targeted than fungi (Table 6.6). Park et al. [255] inoculated the P. citrinum fungus on filefish filets and decontaminated it with oxygen plasma. Norovirus was also targeted for plasma-mediated decontamination. Norovirus is a pathogenic virus that causes vomiting and acute gastroenteritis; consumption of contaminated foods is one of the routes for disease outbreaks [284]. Several studies have shown the efficient decontamination of beef, pork, chicken, oysters, and salmon sashimi contaminated with norovirus of human or murine origin by plasma [15, 47, 122]. As observed in the decontamination of fruits and vegetables, plasma flame or plasma-generated gas generated from various plasma sources was more frequently applied to meats, meat products, and fish than plasma-treated water or liquids (Table 6.6). Interestingly, Qian et al. [275] found that plasma-activated lactic acid was more effective in decreasing microbial load contaminated on chicken drumsticks than plasma-treated water.

Generally, non-thermal plasma treatment, either gaseous plasma or plasma-treated liquid, can deactivate bacteria inoculated or contaminated on meats, meat products, and fish, with an efficiency of 0.1–6.52 log CFU reduction depending on treatment time and conditions. Regarding viruses, over 90% of murine norovirus and hepatitis A virus copy numbers were reduced after plasma treatment of contaminated beef, pork, and chicken [15]. In oysters contaminated with human norovirus, the virucidal effect of plasma was negligible (<1 log copy number/μL) without propidium monoazide pre-treatment and greater (>1 log copy number/μL) with propidium monoazide pre-treatment [47]. Huang et al. [122] found that N2 plasma deactivated the human norovirus in salmon sashimi with an efficiency of 20% reduction in copy number, and the level of norovirus was undetectable after treatment with O2 plasma.

No or minor deterioration in food quality, such as lipid peroxidation, pH, and sensory properties (appearance, color, odor, acceptability), was observed in the majority of studies (Table 6.6). However, Kim et al. [154] demonstrated that sensory quality parameters such as appearance, color, odor, and acceptability were significantly reduced after plasma treatment of bacteria-contaminated pork loin. This indicates that condition tuning or the development of methods for quality control may be necessary for industrial and market applications.

6.4.3 Packaged Foods

Various food products and fresh produce are often distributed as packaged materials in the market and industry. Prevention of microbial contamination during the packaging process can play an important role in ensuring a long shelf life. Thermal treatment is a routine method for sanitation of packaged foods. However, deterioration of food quality has limited the range of applications of thermal sanitation. Fresh produce is more frequently distributed in packaged states in recent markets. The demand for non-thermal sanitation technologies has increased, particularly in the management of packaged foods.

Non-thermal plasma has demonstrated the potential for microbial decontamination of packaged foods over the last decade. A distinguishing point in these studies was that foods were treated with plasma generated inside the package. Recently, Misra et al. [215] reported an excellent review of the application of non-thermal plasma technology to the sanitation of packaged foods. In Table 6.6, studies excluding those mentioned in Misra et al.’s review are indicated. Various designs of plasma systems specialized for in-package treatment have been developed; a package is placed between two electrodes, electrodes are placed on one side of the package, and electrodes are placed inside the package [215]. In most studies, dielectric barrier discharge (DBD) or surface dielectric barrier discharge (SDBD) plasma was used in the treatment of packaged foods (Table 6.2) [215]. Foods inoculated with food poisoning and spoiling bacteria are most frequently targeted for in-package plasma treatment [215], whereas fungi and viruses have rarely been explored [169, 213, 318, 377]. Foods contaminated with natural microflora were also analyzed after in-package plasma treatment [3, 4, 162, 189, 204, 214, 353, 387]. The efficiency of in-package food sanitation using plasma is good; a >1 log reduction in CFU number has been observed in most studies, and complete eradication of microorganisms has been demonstrated in some cases [4, 103, 172, 387, 388].

In-package food quality after plasma treatment is also an important factor to be considered. Most studies have demonstrated that in-package plasma treatment causes minor or no changes in physiological, physical, and sensory properties (Table 6.6) [215]. However, a recent study demonstrated that plasma treatment could result in lipid peroxidation and significant color changes in packaged ham [369].

6.4.4 Processed Foods

Plasma has been actively applied to the sanitation of processed foods such as juice, milk, cheese, pepper powder, insect powder, and snacks (Table 6.6). Non-thermal tools such as ultrasonification, UV, ionizing radiation, and electrical fields have been applied to the sanitation of heat-sensitive foods [274]. Non-thermal plasma is also considered a promising technology that can efficiently remove microbial contamination during food processing and packaging. Liquid foods such as fruit juice and milk have been frequent targets for plasma sanitation, and a greater than 1 log reduction in bacterial CFU number was obtained after plasma treatment (Table 6.6). In various studies, the quality of juices and milk was not significantly affected by plasma (Table 6.6). However, Xu et al. [365] found that direct treatment with 90 kV high voltage atmospheric cold plasma reduced vitamin C content by 22% and pectin methylesterase activity by 74–82% in orange juice. Muhammad et al. [231] showed that DBD air plasma caused a significant reduction in pH, protein content, and peroxidase activity in tiger nut milk, whereas no significant changes in soluble solids and fat contents were observed.

The sanitation of dry foods and powders using plasma resulted in an efficient >1 log reduction in CFUs in most cases (Table 6.6). Bacteria inoculated on sliced cheese were efficiently inactivated in encapsulated or flexible thin-layer DBD plasma systems, and some food qualities such as flavor, overall acceptance, and off-color were significantly affected by plasma [374, 376]. Dry powders, such as onion powder, black pepper powder, and insect powder, were efficiently decontaminated with no dramatic changes in food quality [156, 173, 268]. Several studies have demonstrated that plasma treatment can alter protein solubility and the amount of lipids, chlorophyll a, carotenoids, phycobilin, and total phenolic compounds in wheat flour, insect powder, and algae powder [20, 29, 41]. Particularly, Bahrami et al. [20] observed no significant changes in total aerobic bacterial count or total mould count in wheat flour after treatment with 0.19 and 0.43 W/cm2 air plasma.

6.5 Application of Non-thermal Plasma to Food Quality and Functional Property

Non-thermal plasma has also been used to enhance the quality and functionality of foods and food ingredients (Table 6.6). The quality and nutritional value of fresh produce are investigated together during plasma sanitation to determine whether plasma treatment can affect food quality. Color, texture, pH, proteins, carbohydrates, vitamins, lipids, and antioxidant activity are major properties frequently analyzed in previous studies [249]. These factors are mostly related to the taste, nutritional value, and senescence of the fresh produce. In many studies, plasma treatment did not cause significant damage to the quality of fresh produce. Improvement in antioxidant activity and increase in phenolic content are often observed in lettuce, cut apples, potatoes, peanuts, and grapes [23, 39, 90, 98]. Rinsing with plasma-treated water can improve the color and texture of fresh-cut endives [295]. Plasma can also increase the speed of drying and improve the quality of raisins from fresh grapes [120].

Furthermore, studies have demonstrated that plasma can affect the quality and functionality of food ingredients and processed food products (Table 6.6). The redness of meat can be improved by increasing the amount of nitrite in the meat after plasma treatment [144, 378]. The nutritional value of several herbs, such as fenugreek, pearl millet, and lemon verbena, is also enhanced by plasma. Plasma can facilitate the acquisition of galactomannan from fenugreek, improve the hydration of pearl millet, and elevate the contents of monoterpene hydrocarbons and oxygenated sesquiterpenes in lemon verbena [75, 185, 279]. Moreover, starch structure can be modified by plasma, which can further alter the properties of starch such as solubility, depolymerization, and paste viscosity, making it more suitable for food and non-food industries [43, 314, 361, 384]. Plasma can improve the storage of soybean and peanut oils and the functionality of wheat and soybean proteins [333, 383]. Additionally, plasma can increase depolymerization of inulin for the production of fructooligosaccharides without changing its quality as a food ingredient [85, 236].

6.6 Conclusion and Future Perspectives

Non-thermal atmospheric- and low-pressure plasma are promising tools for several applications, such as microbial decontamination and activation of seed germination and growth, in the food and agriculture industries. However, the mechanisms underlying plasma action, standardization of applied plasma dose, and development of industrial-scale treatments still need for intense further study. The scale addressed in the agriculture and food industries is relatively large compared to that in the medical field, and this should be considered when developing a plasma system. Another future direction in plasma application may be that plasma can be explored to find a potential solution to agricultural and food issues resulted from climatic change. Due to climate change, the current agriculture and food industry is facing a big challenge, and improvement in stress tolerance and storage of fresh produce has received increasing attention as emerging areas wherein plasma can be applied.