Abstract
Soil-borne pathogenic microorganisms are known to cause extensive crop losses. Agrobacterium tumefaciens, a member of the Proteobacteria, causes the neoplastic crown gall disease in plants. Plant protection is mainly based on toxic chemicals that are harmful to the environment. The use of cold atmospheric-pressure plasma is an attractive method for microbial eradication. Its antimicrobial mechanism includes the formation of large quantities of reactive oxygen species (ROS). The advantages of eradicating bacteria using cold plasma are not needed for chemicals, short treatment, and environmental temperatures. This study examined the impact of plasma corona discharge exposure on A. tumefaciens viability, membrane permeability, relative cell size, and ROS formation. The results showed that 90 s of plasma exposure led to a reduction by four orders of magnitude when the initial concentration was 1 × 107 CFU/mL and in a dry environment. When the initial concentration was 1 × 106 CFU/mL, 45 s of exposure resulted in total bacterial eradication. In a liquid environment, in an initial concentration of 2.02 × 106 CFU/mL, there was no complete bacterial eradication even at the most prolonged examined exposure (90 s). The influence of plasma treatment on the membrane permeability of A. tumefaciens, and their possible recovery, were analyzed using flow cytometer analysis using propidium iodide (PI). When the plasma-treated bacteria were suspended in Luria–Bertani (LB) (rich medium), the PI-positive count of the plasma-treated bacteria after two hours was 12 ± 3.9%. At the 24th hour, this percentage was only 1.74 ± 0.6%, as the control (0.7 ± 0.1%). These results may indicate the repair of the plasma-treated bacteria that were suspended in LB. At the 24th hour, the relative cell size of the treated bacteria shifted to the right, to ~3 × 104 forward side scatter (FSC), about 0.5-fold higher than the untreated cells. Measurement of the ROS showed that the intracellular fluorescence of the 90-s plasma-treated cells led to significant fluorescence formation of 32 relative fluorescence units (RFU)/cell (9 × 104 fold, compared to the nontreated cells). This study showed that cold plasma is a useful method for A. tumefaciens eradication. The eradication mechanism involves ROS generation, membrane permeability, and changes in cell size.
Highlights
The bacterial samples with two different initial concentrations of 107 and 106 colony-forming unit (CFU)/mL were centrifuged, and the sediment was suspended in phosphate-buffered saline (PBS)
The results showed that at an initial concentration of 107 CFU/mL, plasma treatment of 30 s led to a minor bacterial reduction from 107 CFU/mL to 2.6 × 106 CFU/mL
Plasma treatment of 10 s led to bacterial reduction of three orders of magnitude from 1.1 × 106 to 3.9 × 103 CFU/mL
Summary
The antimicrobial effect and mechanism of plasma disinfection include the formation of large quantities of reactive oxygen species (ROS), including atomic oxygen (O), hydroxyl radicals (·OH), hydrogen peroxide (H2 O2 ), ozone (O3 ), and singlet oxygen (1 O2 ) [26,27,28], as well as multiple reactive nitrogen species (RNS) such as NO (nitric oxide) and ONOO− (peroxynitrite). These molecules play a significant role in the plasma biocidal process by altering the cell-wall components, the functions and structure of the phospholipid bilayer, the structure of nucleic acids and cellular proteins, gene expressions, and protein synthesis [26,29,30]. This research investigated the effect of plasma corona discharges on the eradication of A. tumefaciens by impacting its viability, membrane permeability, relative cell size, and reactive oxygen formation
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