In the year 2050 it is estimated that the world’s population will have reached 9.7 billion, and worldwide crop production will need to increase two-fold in order to cover the demand by this time (1,2). An effective way to increase crop yield is to minimize losses to pathogenic microorganisms by improving the disease resistance of crop plants. Generally, this is done by selective breeding and the application of disease treatments (fungicides, bactericides). These approaches may, however, have detrimental side effects. For instance, constitutively activated plant defense responses could lead to allocation of plant resources to defense response mechanisms instead of developmental processes and, in the worst case, cause decreased plant growth and yield (3 - 6). Plant pathogens could become resistant to disease treatments and the treatments themselves may pose risks to the environment and consumers in the long-term (7).
A recently emerging alternative to conventional methods for improving plant disease resistance is cold plasma in the form of cold gas and cold solution plasma. Plasma describes matter which partly consists of charged components, ions and electrons, and is considered the fourth state of matter (8).
Cold gas plasma is produced by exposing atmospheric gases to high energy in the form of electrical discharges (9,10,11) or microwaves (12) at atmospheric pressure. This ionizes the gas and generates reactive components like reactive oxygen species (ROS) and reactive nitrogen species (RNS), as well as UV-light, ions and electrons (10,13 - 17). Depending on the utilized gas, nitrous oxide (N2O) and carbon dioxide (CO2) are also produced (11,18). Since the temperature of gas plasma stays around room temperature during its generation, it is called cold gas plasma (19). In nature, gas plasma is found in large quantities in the Earth’s ionosphere (20).
Cold solution plasma or liquid-phase plasma is generated by exposing solutions (hexane (C6H14), sodium acetate (CH3COONa), or sodium sulfate (Na2SO4) solutions for example) to high energy via microwaves, and by channeling air-based cold plasma through the solution, at atmospheric pressure. Plasma activated water (PAW) has also been generated by electrical discharge (18,21,22). Solution plasma also remains at room temperature (21). In nature, PAW in the form of rain is generated during lightning (23).
Significantly, investigations suggest that cold plasma mediates plant disease control, and improves seed germination and plant growth. Cold plasma has also been used for seed sterilization in recent years (10,11,18,21,23 - 25).
Effect of cold plasma on microorganisms
The characteristic of cold plasma to generate reactive species, UV-light, ions and electrons infers the potential to inactivate microorganisms. For this purpose, cold plasma has been tested for microbial decontamination and disease control on plants.
Cold plasma inactivates microorganisms
Takai (2008) produced cold solution plasmas (sodium acetate and sodium sulfate solutions) and showed that they killed Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) in highly concentrated suspensions after 30 seconds of treatment. Likewise, E. coli and S. aureus cell suspensions in water were inactivated within 5 and 15 minutes of continuous application of a nitric oxide (NO)-based plasma (16). Strains of both bacteria are known to cause serious diseases in humans (26,27). PAW, however, inactivated only some spores of the fungus Fusarium fujikuroi (F. fujikuroi) (fungal pathogen of rice (28)) after exposure for 10 minutes (29). Cold argon plasma, on the other hand, effectively killed Fusarium oxysporum (F. oxysporum) spores (causes wilt on several crop plants (30,31)) after 10 minutes of exposure (17).
These results suggest that cold plasma, especially gas plasma, could be utilized to effectively decontaminate solutions such as wastewater (32), but also that certain cold plasmas could be more suited for the inactivation of specific microbes in solution/water.
Cold plasma decontaminates seeds
Seeds can harbor plant pathogens in the seed coat or in biofilms on the surface (33,34) and can cause disease in seedlings once they gain sufficient population density. Seed-borne pathogens are difficult to inactivate due to their location and consequent protection from outside influences. Seeds are commonly treated with insecticides and fungicides, but also with heat and radiation (35,36).
Investigations on the effects of cold plasma on crop seeds indicate a plasma mediated inactivation of microbes. Cold argon and air plasma treatments of rice seeds inactivated seed-borne pathogens and resulted in minor fungal infection of the emerging seedlings. Emerging seedlings of untreated seeds became heavily infected (37).
PAW was found to kill the spores on 80 % of F. fujikuroi infected rice seed. Scanning electron microscopy showed that almost all F. fujikuroi spores were detached from these seeds, contrary to control seeds, where fungal spores were still attached. This suggests that plasma additionally mediates mechanical detachment of spores (29).
Overall, cold plasmas seem to affect seed-borne pathogenic fungi and partly inactivate them. It could be speculated that due to the internal localisation of some microbes (and formation of biofilms), they were protected from cold plasma and survived the treatment.
Cold plasma inactivates harmful microorganisms on fruits and vegetables
Vegetables and fruits, such as lettuce, basil, and tomatoes, can be carriers of human pathogens (38,39,40,41). Cold plasma could be an alternative to commonly used agents, such as organic acids and radiation, to decontaminate fruits and vegetables. E. coli, Salmonella enterica (S. enterica) and Listeria monocytogenes were effectively inactivated by cold air plasma on cherry tomatoes and apples without detrimental effects on the fruits (11,44). On strawberries that have a more uneven surface, however, cold air plasma treatment was less effective, but still reduced bacterial concentrations, without detrimental effects on the fruits (11). Similarly, cold hydrogen peroxide (H2O2) solution plasma effectively inactivated the E. coli strain O157:H7, Salmonella thyphimurium (S. thyphimurium) and Listeria innocua (L. innocua) on tomatoes, baby spinach leaves, tomato stem scars and cantaloupe rinds. All these bacteria are known to cause serious diseases in humans (38,40,42,43,45,46). H2O2 itself, without plasma activation, is utilised as an antimicrobial agent but its effectiveness is considered low (47). On tomatoes and spinach leaves, S. thyphimurium and L. innocua were inactivated much more effectively than on tomato stem scars and cantaloupe rinds. E. coli O157:H7 was only effectively inactivated by cold plasma on tomatoes (48). These results suggest that uneven surfaces, like that of strawberries, could contribute to the survival of some bacteria (11).
Overall, cold gas plasma shows potential in food decontamination by removing food-borne microbes.
Cold plasma as a plant disease control agent
As cold plasma inactivates bacteria and fungi on seeds and fruit/vegetables with seemingly no effect on the fruit/vegetables and seed germination (37), cold plasma was tested in plant disease control.
A single cold helium plasma treatment of Ralstonia solanacearum (causes wilt on several crops (49)) infected tomato plants delayed wilting and slowed disease progress by 25 %, 20 days after gas plasma treatment (10). Similarly, cold argon plasma treatment of F. oxysporum infected tomato plants resulted in an inactivation of F. oxysporum spores. Molecular biological investigations found that gas plasma treatment alone led to the expression of pathogenesis-related (PR) genes. Unfortunately, it was not determined how these PR genes were affected by gas plasma in F. oxysporon infected tomato plants. Nevertheless, surprisingly the PR gene expression changes were observed in the roots and not the leaves that were actually treated with gas plasma. A defense response is usually activated at the site of treatment or systemically.
It was hypothesized that reactive components of cold plasma, such as ROS and RNS, could have entered the cell and acted as signalling molecules (17). ROS and RNS are important intracellular signalling compounds of the plant defense response (50,51,52). These results suggest that cold gas plasma could mediate plant disease control via reactive components and the induction of defense mechanisms.
The effectiveness of cold plasma-mediated inactivation of microorganisms seems to depend on the one hand on the specific characteristics of the microorganisms. Gram-negative bacteria (e.g. S. enterica and E. coli) have thinner outer membranes and are inactivated much faster than Gram-positive bacteria (e.g. L. monocytogenes). It was suggested that the thinner outer membrane allowed for diffusion of reactive components into the bacteria, subsequently killing it. A thicker membrane, however, could present a barrier for cold gas plasma (11). On the other hand, the plant and plant tissue characteristic also seem to influence the inactivation effectiveness of cold gas plasma. The irregular surface of strawberries and cantaloupe rind could shield bacteria from cold gas plasma and contribute to biofilm-formation. Subsequently, microbes are more protected. After all, bacterial inactivation was reduced on irregular surfaces compared to the smooth surfaces (10,48). The hypothesized protection of microbes due to surface differences could also apply to seeds (29,37).
Overall, these results indicate a beneficial effect of cold gas plasma on plant disease control, but the current scientific knowledge on the effects of cold plasma is still at an early stage.
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