Cold plasma in seed, fruit and vegetable decontamination and plant disease control – Part 2

01.09.2017  /  Scienceandmore  /  Category: Plant Biology

This blog post is the continuation of the post “Cold plasma in seed, fruit and vegetable decontamination and plant disease control – Part 1

Cold plasma utilisation in plant diseases control

The application of cold plasma on seeds and fruits/vegetables suggest effective inactivation of bacteria and fungi and at the same time seemingly little effect on the seeds and fruits/vegetables. This combination suggests beneficial effects of cold plasma in plant disease control without detrimental effects on the plant.

A single cold helium plasma treatment of Ralstonia solanacearum (that can cause wilt on several crops (Peeters et al., 2013)) infected tomato plants delayed wilting and also slowed the disease progress by 25 %, 20 days after gas plasma treatment (Jiang et al., 2014). Similarly, cold argon plasma treated tomato plants that were infected with F. oxysporum resulted in an inactivation of F. oxysporum spores. Molecular biological investigations found that gas plasma treatment alone led to the expression of genes that are associated with plant defence responses in tomato. It was, however, not determined how these PR genes were affected in F. oxysporon infected tomato plants that were treated with cold plasma. Surprisingly, this observed expression of these genes was found in the roots and not the leaves that were actually treated with gas plasma. It is rather expected that defence response is activated at the site of treatment or in the whole plant. This result illustrates that the effects of cold plasma and the triggered mechanisms in plants are still unknown.

Nevertheless, it was hypothesised that reactive components of cold plasma such as reactive oxygen species and reactive nitrogen species could have entered the cell and acted as signalling molecules (Panngom et al., 2014). Reactive oxygen and nitrogen species are important intracellular signalling compounds of the plant defence response (Coll et al., 2011; Glazebrook 2005; Shapiguzov et al., 2012). (For the biology nerds: Jiang et al. (2014) found increased PHENYLALANINE AMMONIA-LYASE 1 (PAL1) expression in R. solanacearum infected tomato plants upon cold gas plasma treatment. PAL1 is involved in salicylic acid (SA) biosynthesis, an important signalling component of the plant defence response (Coll et al., 2011; Wildermuth et al., 2001; Fu et al., 2013). Enzyme activity assays for resistance-related plant peroxidase (POD) and polyphenol oxidase (PPO) in R. solanacearum infected tomato plants showed increased activity of both enzymes. PODs generate H2O2 upon pathogen perception, and are involved in defence response signalling and cell wall lignification (Kawano, 2003). PPOs are involved in herbivore and pathogen defence (Constabel and Barbehenn, 2008)). These results suggest that cold gas plasma could mediate plant disease control via reactive components and the induction of plant defence mechanisms.

Summary

It is generally assumed that reactive oxygen species such as hydrogen peroxide (H2O2) and hydroxyl radicals (OH), reactive nitrogen species such as peroxynitrite (ONOO) and nitrite (NO2), and UV-light as cold plasma components contribute to microbial spore and cell inactivation. The impact of these different components, however, seems to vary between the different forms of cold plasma (Jiang et al., 2014; Laroussi and Leipold, 2004; Laroussi, 2009; Oehmigen et al., 2010; Panngom et al., 2014). Ziuzina et al. (2014) challenged the assumption that reactive oxygen and nitrogen species cause the inactivation of microorganisms. Due to the distance of approximately 16 cm between the tomato plants and the centre of the cold plasma production in their experiment, it was argued that reactive oxygen and nitrogen species would most likely react before reaching the samples (Laroussi, 2009). The group found increasing ozone concentration with increasing treatment duration and hypothesised that ozone could act as microbe inactivating agent of cold gas plasma.

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The effectiveness of cold plasma mediated inactivation of microorganisms seems to depend on one hand on the specific characteristics of the microorganisms. S. enterica and E. coli that are Gram-negative bacteria with thinner outer membrane were inactivated much faster than L. monocytogenes a Gram-positive bacterium with a thicker outer membrane. It was suggested that the thinner outer membrane allowed for a diffusion of reactive components into the bacteria, subsequently killing it, whereas the thicker membrane could present a barrier that leads to reduced inactivation by cold gas plasma (Ziuzina et al., 2014). In addition to the microbial characteristics, the plant and plant tissue characteristic seem to influence the inactivation effectiveness of cold gas plasma as well. The irregular surface of strawberries and cantaloupe rind has been hypothesised to provided sites for microbial attachment that shield bacteria from cold gas plasma and contribute to biofilm-formation, subsequently protecting microbes and reducing the inactivation of E. coli, S. enterica and L. monocytogenes, compared to the smooth surface of tomatoes (Ziuzina et al., 2014; Jiang et al., 2017). This hypothesised protection of microbes due to surface differences could also apply to seeds (Kang et al., 2015; Khamsen et al., 2016).

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.

References

Andino, A., and Hanning, I. (2015) Salmonella enterica: Survival, Colonization, and Virulence Differences among Serovars. The Scientific World Journal, 2015, 520179.

Bormashenko, E., Grynyov, R., Bormashenko, Y., & Drori, E. (2012) Cold Radiofrequency Plasma Treatment Modifies Wettability and Germination Speed of Plant Seeds. Scientific Reports. 2, 741.

Bourke, P., Zuizina, D., Han, L., Cullen, P.J., Gilmore, B.F. (2017) Microbiological Interactions with Cold Plasma. J Appl Microbiol. doi: 10.1111/jam.13429. [Epub ahead of print]

Bruggeman, P. and Leys, C. (2009) Non-thermal plasmas in and in contact with liquids. J. Phys. D. Appl. Phys. 42, 053001

Carter, L.L., Leslie, J.F., Webster, R.K. (2008) Population structure of Fusarium fujikuroi from California rice and water grass. Phytopathology. 98(9), 992-8.

Coll, N.S., Epple, P., Dangl, J.L. (2011): Programmed cell death in the plant immune system. Cell Death and Differentiation, 18, 1247-56.

Constabel, C.P., and Barbehenn, R. (2008) Defensive Roles of Polyphenol Oxidase in Plants in Induced Plant Resistance to Herbivory (ed. Schaller, A.), 253-69. Springer Science+Business Media B.V.

Danhorn, T. and Fuqua, C. (2007) Biofilm Formation by Plant-Associated Bacteria. Annu. Rev. Microbiol.  61, 401-22.

Denancé, N., Sánchez-Vallet, A., Goffner, D., Molina, A. (2013) Disease resistance or growth: the role of plant hormones in balancing immune responses and fitness costs. Front Plant Sci., 4, 155.

Di Pietro, A.D., Madrid, M.P., Caracuel, Z., Delgado-Jarana, J., Roncero, M.I.G. (2003) Fusarium oxysporum: exploring the molecular arsenal of a vascular wilt fungus. Mol Plant Pathol 4, 315-25.

El-Sayed, W. S., Ouf, S. A., & Mohamed, A.-A. H. (2015) Deterioration to extinction of wastewater bacteria by non-thermal atmospheric pressure air plasma as assessed by 16S rDNA-DGGE fingerprinting. Frontiers in Microbiology, 6, 1098.

Engwall, E., Eriksson, A.I., Cully,C.M., André, M., Torbert, R., Vaith, H. (2009) Earth’s ionospheric outflow dominated by hidden cold plasma. Nature Geoscience. 2, 24-7.

Fravel, D., Olivain, C. and Alabouvette, C. (2003) Fusarium oxysporum and Its Biocontrol. New Phytologist, 157, 493-502.

Fu, Z.Q. and Dong X. (2013) Systemic Acquired Resistance: Turning Local Infection into Global Defence. Annu Rev Plant Biol., 64, 839-63.

Glazebrook, J. (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens. Annu Rev Phytopathol., 43, 205-27.

Goossens, M. (2003) An Introduction to Plasma Astrophysics and Magnetohydrodynamics., p25. Springer Science & Business Media. NL

Heidel, A.J., Clarke, J.D., Antonovics, J., Dong, X.N. (2004) Fitness costs of mutations affecting the systemic acquired resistance pathway in Arabidopsis thaliana. Genetics, 168, 2197-206.

Heil, M., Hilpert, A., Kaiser, W., and Linsenmair, K.E. (2000). Reduced growth and seed set following chemical induction of pathogen defence: does systemic acquired resistance (SAR) incur allocation costs? J Ecol., 88, 645-54.

Heinlin, J., Morfill, G., Landthaler, M., Stolz, W., Isbary, G,. et al. (2010) Plasma medicine: possible applications in dermatology. J Dtsch Dermatol Ges. 8, 968-76.

Huot, B., Yao, J., Montgomery, B.L., He, S.Y. (2014) Growth–Defense Tradeoffs in Plants: A Balancing Act to Optimize Fitness. Molecular Plant, 7, 1267-87.

Jiang, J., Lu, YX., Li, J., Li, L., He, X., Shao, H., Dong, Y. (2014) Effect of Seed Treatment by Cold Plasma on the Resistance of Tomato to Ralstonia solanacearum (Bacterial Wilt). PLoS ONE 9(5), e97753.

Jiang, Y., Sokorai, K., Pyrgiotakis, G., Demokritou, P., Li, X., Mukhopadhyay, S., Jin, T., Fan, X. (2017) Cold plasma-activated hydrogen peroxide aerosol inactivates Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria innocua and maintains quality of grape tomato, spinach and cantaloupe. Int J Food Microbiol. 249, 53-60.

Jørgensen, L.N., van den Bosch, F., Oliver, R.P., Heick, T.M., Paveley, N. (2017) Targeting Fungicide Inputs According to Need. Annu Rev Phytopathol. doi: 10.1146/annurev-phyto-080516-035357. [Epub ahead of print]

Kang, M. H., Pengkit, A., Choi, K., Jeon, S. S., Choi, H. W., Shin, D. B., Choi, E.H., Uhm, H.S., Park, G. (2015) Differential Inactivation of Fungal Spores in Water and on Seeds by Ozone and Arc Discharge Plasma. PLoS ONE, 10(9), e0139263.

Kawano, T (2003) Roles of the reactive oxygen species-generating peroxidase reactions in plant defense and growth induction. Plant Cell Rep. 21(9), 829-37.

Khamsen, N., Onwimol, D., Teerakawanich, N., Dechanupaprittha, S., Kanokbannakorn, W., Hongesombut, K., Srisonphan, S. (2016) Rice (Oryza sativa L.) Seed Sterilization and Germination Enhancement via Atmospheric Hybrid Nonthermal Discharge Plasma. ACS Appl Mater Interfaces. 8(30), 19268-75.

Kim, J.H. and Min, S.C. (2017) Microwave-powered cold plasma treatment for improving microbiological safety of cherry tomato against Salmonella. Postharvest Biology and Technology. 127, 21-6.

Laroussi, M. (2009) Low-temperature plasmas for medicine? Plasma Science. IEEE Trans. Plasma Sci. 37, 714-25.

Laroussi, M. and Leipold, F. (2004) Evaluation of the roles of reactive species, heat, and UV radiation in the inactivation of bacterial cells by air plasmas at atmospheric pressure. Int. J. Mass Spectrom. 233, 81-6.

Leenders, P. (2015) Plasma activated water [Presentation]. TED Talk Arnheim. 07.10.2015

Lim, J.Y., Yoon, J.W., Hovde, C.J. (2010) A Brief Overview of Escherichia coli O157:H7 and Its Plasmid O157. Journal of Microbiology and Biotechnology, 20(1), 5-14.

Niemira, B.A. and Sites, J. (2008) Cold plasma inactivates Salmonella Stanley and Escherichia coli O157:H7 inoculated on golden delicious apples. J Food Prot. 71(7), 1357-65.

Oehmigen, K., Hähnel, M., Brandenburg, R., Wilke, C., Weltmann, K.-D., von Woedtke, T. (2010) The Role of Acidification for Antimicrobial Activity of Atmospheric Pressure Plasma in Liquids. Plasma Processes and Polymers. 7( 3-4), 250-7.

Olaimat, A.N., Holley, R.A. (2012) Factors influencing the microbial safety of fresh produce: a review. Food Microbiol. 32, 1e19.

Ölmez, H., Kretzschmar, U. (2009) Potential alternative disinfection methods for organic fresh-cut industry for minimizing water consumption and environmental impact. LWT Food Sci. Technol. 42, 686-93.

Panngom, K., Lee, S. H., Park, D. H., Sim, G. B., Kim, Y. H., Uhm, H. S., et al. (2014) Non-Thermal Plasma Treatment Diminishes Fungal Viability and Up-Regulates Resistance Genes in a Plant Host. PLoS ONE, 9(6), e99300.

Peeters, N., Guidot, A., Vailleau, F., Valls, M. (2013) Ralstonia solanacearum, a widespread bacterial plant pathogen in the post-genomic era. Mol Plant Pathol. 14(7), 651-62.

Perrin, M., Bemer, M., & Delamare, C. (2003) Fatal Case of Listeria innocua Bacteremia. Journal of Clinical Microbiology, 41(11), 5308-9.

Ramaswamy, V., Cresence, V.M., Rejitha, J.S., Lekshmi, M.U., Dharsana, K.S., Prasad, S.P., Vijila, H.M. (2007) Listeria – review of epidemiology and pathogenesis. J. Microbiol. Immunol. Infect. 40(1), 4-13.

Rangel, J.M., Sparling, P.H., Crowe, C., Griffin, P.M., Swerdlow, D.L. (2005) Epidemiology of Escherichia coli O157:H7 outbreaks, United States, 1982e2002. Emerg. Infect. Dis. 11, 603e609.

Raybaudi-Massilia, R.M., Mosqueda-Melgar, J., Soliva-Fortuny, R., Martin-Belloso, O. (2009) Control of pathogenic and spoilage microorganisms in fresh-cut fruits and fruit juices by traditional and alternative natural antimicrobials. Compr. Rev. Food Sci. Food Saf. 8, 157e180.

Sera, B., Spatenka, P., Sery, M., Vrchotova, N., Hruskova, I. (2010) Influence of Plasma Treatment on Wheat and Oat Germination and Early Growth. IEEE Transactions on Plasma Science. 38(10), 2963-8.

Shapiguzov, A., Vainonen, J.P., Wrzaczek, M., Kangasjärvi, J. (2012) ROS-talk – how the apoplast, the chloroplast, and the nucleus get the message through. Front Plant Sci., 3, 292.

Sharma, K.K., Singh, U.S., Sharma, P., Kumar, A., Sharma, L. (2015) Seed treatments for sustainable agriculture-A review. Journal of Applied and Natural Science. 7(1), 521-39.

Sivachandiran, L. and Khacef, A. (2017) Enhanced seed germination and plant growth by atmospheric pressure cold air plasma: combined effect of seed and water treatment. RSC. 7, 1822-32.

Stephen, J., Amin, I., Douce, G.R. (1993) Experimental Salmonella typhimurium-induced gastroenteritis. In Biology of Salmonella (eds. Cabello, F., Hormaeche, C., Mastreoni, P., Bonina, L.,), 199-209. Plenum Press, N.Y.

Takai, O. (2008) Solution plasma processing (SPP). Pure Appl. Chem., 80(9), 2003-11.

Tilman, D., Balzer, C., Hill, J., & Befort, B. L. (2011) Global food demand and the sustainable intensification of agriculture. Proceedings of the National Academy of Sciences of the United States of America, 108(50), 20260-4.

Tong, S.Y., Davis, J.S., Eichenberger, E., Holland, T.L., Fowler, V.G. (2015) Staphylococcus aureus infections: epidemiology, pathophysiology, clinical manifestations, and management. Clinical Microbiology Reviews. 28 (3), 603-61.

Toval, F., Schiller, R., Meisen, I., Putze, J., Kouzel, I. U., Zhang, W., et al. (2014) Characterization of Urinary Tract Infection-Associated Shiga Toxin-Producing Escherichia coli. Infection and Immunity, 82(11), 4631-42.

Tsedaley, B. (2015) Review on Seed Health Tests and Detection Methods of Seedborne Diseases. Journal of Biology, Agriculture and Healthcare. 5(5), 176-84.

United Nations, Department of Economic and Social Affairs, Population Division (2015) World Population Prospects: The 2015 Revision, Key Findings and Advance Tables. Working Paper No. ESA/P/WP.241.

Volin J.C., Denes, F.S., Young, R.A., and Park, S.M.T. (2000) Modification of seed germination performance through cold plasma chemistry technology. Crop. Sci 40, 1706-18.

Wang, H., Zhou, B., Feng, H. (2012) Surface characteristics of fresh produce and their impact on attachment and removal of human pathogens. Produce contamination. In Decontamination of Fresh and Minimally Processed Produce (ed. by Gomez-Lopez, V.M.), 43-55. Wiley-Blackwell Publishing, USA.

Ziuzina, D., Patil, S. Cullen, P.J., Keener, K.M., Bourke, P. (2014) Atmospheric cold plasma inactivation of Escherichia coli, Salmonella enterica serovar Typhimurium and Listeria monocytogenes inoculated on fresh produce. Food Microbiology 42, 109e116.

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