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Article

Comparative Analysis of Plasma Technologies for Plant Growth Enhancement and Microbial Control: A Systematic Optimization Study

by
Binoop Mohan
1,†,
Chandrima Karthik
2,†,
Chippy Pushpangathan
3,
Karolina M. Pajerowska-Mukhtar
4,
Vinoy Thomas
2 and
M Shahid Mukhtar
1,*
1
Department of Genetics & Biochemistry, Clemson University, 105 Collings St., Clemson, SC 29634, USA
2
Department of Mechanical and Materials Engineering, University of Alabama at Birmingham (UAB), Birmingham, AL 35294, USA
3
Department of Geography, Central University of Karnataka, Aland Road, Kadaganchi 585367, Karnataka, India
4
Department of Biological Sciences, Clemson University, 132 Long Hall, Clemson, SC 29634, USA
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Int. J. Plant Biol. 2025, 16(3), 104; https://doi.org/10.3390/ijpb16030104
Submission received: 9 July 2025 / Revised: 30 August 2025 / Accepted: 1 September 2025 / Published: 5 September 2025
(This article belongs to the Section Plant–Microorganisms Interactions)
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Abstract

The application of plasma technology in agriculture has emerged as a promising approach to enhance plant health and manage microbial interactions, offering potential solutions for sustainable crop production and disease control. This study contributes to this field by exploring the effects of plasma treatments on plant physiology and microbial dynamics, with a focus on their potential to improve agricultural outcomes. This investigation aims to systematically determine optimal plasma seed treatment parameters for enhancing plant vigor and promoting beneficial microbial associations while minimizing pathogenic interactions in Arabidopsis thaliana. This study focuses on understanding the effects of various plasma treatments on chlorophyll content, root length, microbial growth, and microbial quantification in plants and microbes. The treatments involve the use of an atmospheric jet plasma handheld device, a globe plasma, and a glow discharge plasma chamber with air and argon. These treatments were applied for varying time durations from 10 s to 5 min. The results demonstrated that the globe plasma treatment for 1 min significantly enhanced chlorophyll a extraction and root length, outperforming the other treatments. Additionally, the study examined the impact of plasma on plant–microbe interactions to assess whether plasma treatments affect beneficial microbes. Plasma treatments showed minimal impact on most beneficial microbe activity, though species-specific sensitivities were observed, with Pseudomonas cedrina showing moderate growth inhibition, revealing no significant disruption to their activity. The microbial quantification assays indicated that the globe plasma treatment effectively reduced microbial counts, while combined treatment with plant and microbe plasma together did not yield significant changes. Additionally, the chlorophyll estimation of plasma-treated samples indicated that the globe plasma and atmospheric jet plasma treatments were effective in enhancing chlorophyll content, whereas the combined treatment with both plant and microbe plasma did not yield significant changes. These findings suggest that plasma treatments, especially the globe plasma, are effective in improving plant health and controlling microbial activity. Future research should focus on optimizing plasma conditions, exploring the influence of plasma parameters and the underlying mechanisms, and expanding the scope to include a wider range of plant species and microbial strains to maximize the benefits of plasma technology in agriculture.

1. Introduction

The global agricultural sector faces mounting challenges in sustainably meeting food security demands while minimizing environmental impacts. Innovative approaches that enhance plant productivity and disease resistance without chemical inputs represent a critical frontier in agricultural advancement [1,2]. The agricultural sector has witnessed significant transformations through plasma technology applications, particularly in understanding plant–microbe dynamics [3]. Low-temperature plasma (LTP), or cold atmospheric plasma, treatment has revolutionized approaches to enhance plant vigor and pathogen resistance by leveraging its distinctive physicochemical attributes [4]. Within this context, the genetic model Arabidopsis thaliana serves as an exceptional experimental system for exploring plasma-mediated alterations in plant vigor and plant–microbe relationships, owing to its thoroughly documented genome and rich genetic resources [5,6]. Plasma treatment generates reactive oxygen species such as ozone and hydroxyl radicals, along with reactive nitrogen species and peroxynitrite. These reactive species play dual roles in plant systems: they activate antioxidant defense mechanisms and function as signaling molecules that modulate key phytohormonal pathways, including those involving salicylic acid and jasmonic acid [7]. This signaling cascade is crucial for orchestrating plant immune responses and influences the recruitment and activity of rhizosphere microorganisms. Furthermore, plasma exposure alters the composition of root exudates, particularly organic acids and secondary metabolites, thereby reshaping the chemical dialogue between plant roots and soil microbial communities [8].
Plant health and development critically depend on interactions with microorganisms, orchestrated through intricate signaling networks and metabolic exchanges [9]. External interventions can substantially reshape these molecular conversations [10]. These interactions form the foundation of plant nutrition, stress tolerance, and disease resistance [11,12]. The application of plasma generates an array of bioactive molecules, including reactive oxygen and nitrogen species, presenting novel opportunities for modulating plant–microbe relationships [13]. Nevertheless, despite growing interest in this field, our understanding of how plasma treatment influences biological processes, including photosynthesis and plant defense mechanisms, and shapes microbial communities requires deeper investigation, particularly regarding optimization of treatment parameters [14,15]. Recent advancements in plasma agriculture have demonstrated significant progress in non-thermal plasma applications for seed germination and plant growth [16], systematic analyses of plasma agrotechnology’s identifying key achievements and implementation obstacles [17], and sustainable plasma processing developments emphasizing residue-free agricultural applications [18], establishing plasma technology as a promising frontier for addressing contemporary agricultural challenges and food security demands.
LTP systems can improve the germination rates, seedling vigor, seed surface alterations, microbial growth, etc., with the help of the generation of reactive oxygen and nitrogen species (RONS) at or near ambient temperatures, which minimizes thermal damage. Glow discharge plasma systems utilize a gas at low pressure between two electrodes to create a self-sustaining plasma that emits a characteristic colored glow. The color of the plasma depends on the gas used. The atmospheric pressure plasma is formed at or near ambient pressure with air as the medium of gas. The jet device directs a stream of plasma (ionized gas) onto the targeting surface for the functionalization. A plasma globe is a glass sphere filled with a mixture of inert gases, such as neon and argon, with a partial vacuum. Inside the globe, a Tesla coil generates high-frequency plasma that extends toward the inner surface of the globe [19]. Plasma seed treatment, involving the exposure of dormant seeds to non-thermal plasma prior to germination, has emerged as a promising pre-sowing technique for enhancing subsequent plant performance and modifying plant–microbe interactions throughout the plant life cycle. Our experimental design incorporated four plasma systems: dual glow discharge configurations utilizing air and argon, atmospheric plasma jet, and globe discharge plasma filled with a mixture of the noble gases neon, krypton, and xenon. The plasma treatment was conducted at various times (10 s, 30 s, 1 min, and 5 min). This methodical approach enables comparative assessment of plasma variants and identification of optimal exposure periods for enhancing plant–microbe relationships [20]. This systematic approach addresses a critical need for standardization in this emerging field [21].
Research findings have revealed plasma treatment’s capacity to modify plant surface properties and stimulate defense pathways, while restructuring microbial populations. Recent work by Panka et al. [22] demonstrated improved seed germination and disease resistance following plasma exposure. Additionally, Tamosiune et al. [23] documented substantial shifts in root-associated bacterial communities post-treatment. These observations highlight the sophisticated relationship between plasma-induced changes and plant–microbe interactions. This investigation aims to systematically determine optimal plasma parameters for plant vigor and promoting beneficial microbial associations while minimizing pathogenic interactions [24]. Recent developments in plasma agriculture have demonstrated its potential to modulate plant physiology and microbial dynamics through reactive species, yet standardized protocols and mechanistic insights remain limited, underscoring the need for systematic studies to optimize treatment parameters and broaden applicability across diverse plant–microbe systems. Specifically, we focused on evaluating changes in chlorophyll content, root development, rhizosphere microbial composition, and microbial growth dynamics following various plasma treatments. This comprehensive approach provides insights into both plant physiological responses and microbiome effects, advancing our understanding of plasma technology as a sustainable intervention in agricultural systems [25,26].

2. Results

2.1. Plasma Treatment and Its Influence on Plant Chlorophyll Contents

We observed that our MS A3 glow discharge treatment using air for 1 min yielded a w/w of Chl a of approximately 0.025 mg/g, marking a substantial improvement over shorter durations (Figure 1a–c). Our MS B3 atmospheric jet treatment for 1 min resulted in a w/w of Chl a around 0.015 mg/g, reflecting a moderate enhancement. The MS C3 glow discharge using argon for 1 min produced a w/w of Chl a of about 0.02 mg/g, indicating a notable boost. Meanwhile, our MS D3 globe plasma treatment for 1 min achieved a w/w of Chl a of approximately 0.03 mg/g, emerging as the most successful among all treatments and demonstrating the most effective Chl a extraction. In Figure 1d, we present results for the 5-min treatments, where our MS A4 glow discharge using air reached a w/w of Chl a of about 0.018 mg/g, MS B4 atmospheric jet yielded a w/w of Chl a around 0.012 mg/g, MS C4 glow discharge using argon achieved a w/w of Chl a of approximately 0.016 mg/g, and MS D4 globe plasma recorded a w/w of Chl a of about 0.02 mg/g. From these findings, we conclude that our globe plasma treatment for 1 min (MS D3) was the most effective in enhancing Chl a content, achieving the highest w/w of 0.03 mg/g, followed by the argon plasma in the chamber (MS C3) at 0.02 mg/g and air plasma in the chamber (MS A3) at 0.025 mg/g. For the 5-min treatments, the globe plasma (MS D4) remained the most effective, though its extraction efficiency was slightly lower, at 0.02 mg/g, compared to that of the 1-min treatment. We observed that shorter, optimized exposure times, particularly with the globe plasma, maximized Chl a extraction. These results highlight the potential of the globe plasma as a superior method to provide a foundation for optimizing plasma-based techniques in future applications.

2.2. Effect of Plasma Treatment on the Growth of Arabidopsis Roots

The significant data points from the root length measurements are as follows (Figure 2). MS A3 glow discharge using air in chamber for 1 min yielded a primary root length of approximately 120 mm, indicating a substantial increase compared to the control sample; MS B3 atmospheric jet for 1 min yielded a primary root length of around 100 mm, showing a moderate increase; and MS D3 globe plasma for 1 min yielded a primary root length of about 140 mm, the highest among the treatments, indicating the most effective enhancement in root growth. The root images in Figure 3 provide a visual comparison of the root sizes for these significant results against the control sample MS COL3, which had a primary root length of approximately 80 mm. We found that the globe plasma treatment for 1 min (MS D3) resulted in the longest roots, followed by the air plasma in the chamber (MS A3) and the atmospheric jet plasma (MS B3) (Figure 3a–d). These results suggest that the globe plasma treatment was the most effective in promoting root growth, with the air plasma and atmospheric jet plasma also showing positive effects compared to the control.

2.3. Plasma Treatment and Its Impact on Microbial Growth

The data representing the growth curves for both treated and untreated samples were plotted based on OD600nm measurements. The results indicate that the untreated A1 samples exhibited a steady increase in growth, reaching a peak OD600nm of approximately 2.5 at 12 h. In contrast, the treated A1 samples showed a slightly lower growth rate, with a peak OD600nm of around 2.0 at the same time point. For the O12 samples, the untreated group displayed a rapid growth phase, peaking at an OD600nm of about 3.0 at 10 h, while the treated O12 samples had a reduced growth rate, with a peak OD600nm of approximately 2.5 at 10 h. These findings suggest that the glow plasma air treatment had a noticeable impact on the growth dynamics of both microbes, with a more pronounced effect on Pseudomonas cedrina O12.

2.4. Microbial Quantification to Validate the Influence of Plasma on Plant–Microbe Interaction

The results of the plant plasma treatment and microbial quantification assay are presented in Figure 4. Figure 4 shows the microbial quantification from the plasma-treated plant and microbe samples, and looking at the various plasma exposure time points and comparing the values to those for the control samples, it can be clearly seen that the exposure to both plant and microbe plasma treatment did not significantly improve the bacterial growth. The microbial quantification for plant plasma-treated samples is shown in Figure 4. The significant data points include that MA3 glow discharge using air for 1 min showed a notable increase in microbial count compared to the control sample MCOL3, indicating effective microbial growth; MB3 atmospheric jet plasma for 1 min demonstrated a moderate increase in microbial count; MC3 glow discharge using argon for 1 min exhibited a significant increase in microbial count, similar to MA3; and MD3 globe plasma for 1 min had the most substantial increase in microbial count, indicating the highest efficacy in microbial growth among the treatments. Figure 4 shows the microbial quantification from plasma-treated plant and microbe samples, and looking at the various plasma exposure time points and comparing the values to the control samples, it can be clearly seen that exposure to both plant and microbe plasma treatment did not significantly improve the bacterial growth.

2.5. Chlorophyll Estimation to Validate the Influence of Plasma on Plant–Microbe Interaction

The results of the chlorophyll estimation for plant plasma-treated samples and plant and microbe plasma-treated samples are presented in Figure 5. Chlorophyll levels were measured using spectrophotometry, with each sample analyzed in triplicate. Statistical comparisons between individual groups were made using unpaired t-tests, while one-way ANOVA was applied to evaluate differences across multiple groups. We found that for chlorophyll estimation for plant plasma-treated samples, the significant results were obtained for MB3 atmospheric jet for 1 min and MD3 globe plasma for 1 min. The MB3 sample exhibited a notable increase in chlorophyll content compared to the control sample MCOL3, indicating effective chlorophyll enhancement. Similarly, the MD3 sample showed a substantial increase in chlorophyll content, the highest among the treatments, suggesting that the globe plasma treatment was the most effective in enhancing chlorophyll levels. Figure 5 presents the chlorophyll estimation for samples treated with both plant and microbe plasma. However, this figure does not show any significant changes in chlorophyll content compared to the control samples. The reduced chlorophyll content observed in 5-min plasma treatments (MS D4) compared to 1-min treatments (MS D3) can be attributed to dose-dependent cellular stress caused by prolonged plasma exposure. These results suggest that while the globe plasma and atmospheric jet plasma treatments were effective in enhancing chlorophyll content in plant plasma-treated samples, the combined treatment with both plant and microbe plasma did not yield significant changes.

3. Discussion

The comprehensive analysis of the effects of plasma treatments on chlorophyll content, root length, microbial growth, and microbial quantification provides valuable insights into the efficacy of different plasma conditions and reveals potential mechanisms underlying plasma-mediated plant–microbe interactions. Our findings demonstrate distinct effects of plasma type, gas composition, and exposure duration on both plant physiology and microbial dynamics, suggesting multiple pathways through which plasma technologies might be optimized for agricultural applications [27,28]. The initial chlorophyll estimation revealed that the globe plasma treatment for 1 min (MS D3) was the most effective in enhancing Chl a extraction, followed by the argon plasma in the chamber (MS C3) and the air plasma in the chamber (MS A3) [29]. This trend was consistent across various exposure times, with the globe plasma treatment consistently showing the highest chlorophyll content [29,30]. The enhanced chlorophyll content suggests improved photosynthetic capacity, potentially mediated through plasma-induced changes in plant metabolism or nutrient uptake efficiency. The differential effects between plasma types indicate that specific plasma characteristics—possibly related to the composition and energy of reactive species generated—play crucial roles in determining physiological outcomes [31].
In terms of root length, the results indicated that the globe plasma treatment for 1 min (MS D3) resulted in the longest roots, followed by the air plasma in the chamber (MS A3) and the atmospheric jet plasma (MS B3) [32]. The control sample (MS COL3) had significantly shorter roots compared to the treated samples, highlighting the positive impact of plasma treatments on root growth [32,33]. Enhanced root development has profound implications for plant nutrient acquisition, water uptake, and overall stress resilience. The correlation between improved root architecture and specific plasma treatments suggests potential applications in improving crop establishment and resilience, particularly under challenging growth conditions [34,35].
The microbial plasma treatment results demonstrated that the glow plasma air treatment had a noticeable impact on the growth dynamics of both ACC deaminase-positive microbes, A1 (Bacillus cereus) and O12 (Pseudomonas cedrina) [24]. The treated samples showed a reduced growth rate compared to that of the untreated samples, with a more pronounced effect on O12. This suggests that the plasma treatment moderately inhibits microbial growth, which could be beneficial for applications requiring controlled microbial activity [24,36]. The species-specific response observed between Bacillus and Pseudomonas strains indicates that plasma sensitivity may vary across microbial taxa, potentially related to differences in cell wall composition, stress response mechanisms, or metabolic adaptation capabilities. This differential susceptibility could be leveraged to selectively modulate microbiome composition in agricultural settings [37,38].
The microbial quantification assay for plant plasma-treated samples showed that the globe plasma treatment for 1 min (MD3) was the most effective in reducing microbial counts, followed by the argon plasma in the chamber (MC3) and the air plasma in the chamber (MA3) [37]. The atmospheric jet plasma (MB3) also showed positive effects compared to the control samples (MCOL3). However, the combined treatment with both plant and microbe plasma did not show significant changes in microbial quantification compared to the control samples (MT COL3) [24]. This unexpected result with the combined treatment suggests complex interaction effects that warrant further investigation. Possible explanations include plasma-induced changes in plant surface properties, altered root exudate profiles, or induced resistance mechanisms that counteract the direct antimicrobial effects observed with isolated treatments [39,40]. Plant-beneficial microbes tend to increase in number during plasma treatment, but the extent of plasma treatment is critical in determining the impact of plasma on microbial growth. Plasma treatment may improve a plant’s capacity to attract and sustain beneficial microbes by enhancing root secretions or activating immune responses that support symbiotic interactions.
Finally, the chlorophyll estimation for plant plasma-treated samples indicated that the globe plasma (MD3) and atmospheric jet plasma (MB3) treatments were effective in enhancing chlorophyll content [29]. In contrast, the combined treatment with both plant and microbe plasma did not yield significant changes in chlorophyll content compared to that of the control samples [29]. This observation parallels the microbial quantification findings, suggesting coordinated responses across multiple physiological parameters when both plants and microbes are simultaneously exposed to plasma treatment [22]. The simultaneous increase in chlorophyll content and root development observed after plasma treatment indicates a synergistic physiological interaction that enhances overall plant performance. Improved root systems boost the uptake of key nutrients, such as nitrogen, magnesium, and iron, that are vital for chlorophyll synthesis. In turn, the resulting rise in photosynthetic efficiency supplies greater carbohydrate reserves, which support further root expansion by increasing carbon allocation to below-ground tissues. These findings collectively suggest that plasma treatments, particularly the globe plasma, are effective in enhancing chlorophyll content and root growth while also exhibiting microbial suppression capabilities.
The differential effects observed in combined plant and microbe treatments highlight the complexity of interactions and the need for further research to optimize plasma treatment conditions for various applications [24,30,33,36,37]. The duration-dependent effects observed across treatments indicate critical exposure thresholds that must be carefully calibrated to achieve desired outcomes without triggering deleterious responses [41]. Several limitations of the current study should be acknowledged. The experiments were conducted under controlled laboratory conditions with a model plant system, and results may differ in field settings or with crop species. Additionally, while we examined key physiological parameters, the molecular mechanisms underlying the observed effects remain to be elucidated. The microbiome analysis was also limited to specific bacterial strains rather than comprehensive community profiling, which might reveal broader ecological shifts following plasma treatment [42,43]. Additionally, while optical emission spectroscopy confirmed the presence of reactive oxygen and nitrogen species (RONS), their concentrations were not quantified in this study. Future work should incorporate quantitative diagnostics to correlate specific RONS levels with biological responses in plant and microbial systems. Quantitative analysis of these species is essential to establish dose–response relationships and understand the thresholds for beneficial versus harmful effects. Plasma-generated RONS are known to influence seed physiology by modifying the seed coat, enhancing water uptake, and activating signaling pathways related to germination and stress responses.

4. Materials and Methods

4.1. Characteristics and Surface Sterilization of Arabidopsis Seeds

Arabidopsis thaliana Col-0 seeds possess several characteristics that facilitate effective plasma treatment. The thin seed coat, 15–25 μm, and high-water permeability enable efficient penetration of plasma-generated reactive oxygen and nitrogen species. Seeds measuring approximately 0.5 × 0.3 × 0.2 mm provide optimal surface-to-volume ratios for uniform treatment exposure, while the reticulated surface structure maximizes contact area with plasma species. To prepare seed sterilization buffer, mix 50 µL of Triton 10X solution, 700 µL of absolute ethanol, and 3 mL of sterile distilled water gently to get a homogenous solution. Weigh around 100 mg of fresh Arabidopsis seeds and transfer them to a non-sticky 1.5 mL Eppendorf tube (Eppendorf North America, Inc., Enfield, CT, USA) [44]. Start by adding 1 mL of the sterilization buffer into the Eppendorf tube. Transfer the tube to a vortexing machine (Fisher Scientific, Thermo Fisher Scientific, Inc., Waltham, MA, USA) and gently vortex the mixture at a minimal speed to prevent seed damage. Centrifuge the sample at 12,000 rpm for 1–2 min maximum to suspend the seeds. Gently rinse the sterilization buffer by tapping or removing it using a sterile pipette without disturbing the seeds. Add sterile distilled water to the tube containing seeds and vortex well, and centrifuge it at 12,000 rpm for 1–2 min. Continue the rinsing step 3–5 times to ensure that the seeds are free from any traces of the sterilization buffer [45]. Once the steps are completed, the seeds can be suspended in sterile distilled water until they are prepared for the subsequent experimental procedures.

4.2. Estimation of Chlorophyll Content

We conducted a study to evaluate the effects of various plasma treatments on chlorophyll a (Chl a) content, as presented across multiple figures. We applied different plasma conditions, including air and argon in a glow discharge plasma chamber, an atmospheric jet plasma handheld device, and a globe plasma, with treatment durations of 10 s, 30 s, 1 min, and 5 min. Each experiment was conducted three times. Data were analyzed using one-way ANOVA, followed by Tukey’s multiple-comparison test. Results are expressed as means, with statistical significance defined as p < 0.05. We illustrated the weight-to-weight ratio (w/w) of Chl a per gram for each treatment, noting significant increases with longer exposure times. Entire leaf samples from 2-week-old Arabidopsis plants are taken and weighed. The chlorophyll content is compared based on the weight of the leaf sample. The samples are transferred to a 2 mL Eppendorf tube, frozen in a liquid nitrogen-containing cylinder, and homogenized for 30 s, followed by dipping it in the liquid nitrogen and continuing to evenly homogenize the samples. One milliliter of 100% ethanol is added to each tube and kept at 4 °C until the leaves lose their green color. Two hundred microliters of the supernatant was transferred to a 96-well plate, and the readings for chlorophyll measurement were done at 652 and 665 nm wavelengths. All calculations for chlorophyll estimation and quantification were performed according to standard protocol [46,47].

4.3. Measurement of Arabidopsis Root Length

Arabidopsis seeds previously sterilized were used in this experiment. Seeds were exposed to different treatment conditions as mentioned for plasma optimization treatment, and untreated seed samples were initially sown on MS Murashige and Skoog agar plates and transferred to the UAB plant growth facility with optimum growth parameters, including 12-h light and 12-h dark phases with temperature set at 21 °C and light intensity and humidity set respectively at 100 μmoL/m2/s and 45%. The seeds were left to grow in the chamber for 12 days, and later the seedlings were moved to a clear white sheet with proper labelling. Special care was taken to prevent the roots from rolling back and to reduce root damage while handling. We examined the effects of different plasma conditions on root length, including air and argon in a glow discharge plasma chamber, an atmospheric jet plasma handheld device, and a globe plasma, applied for durations of 10 s, 30 s, 1 min, and 5 min. Root length measurements were performed on 24 seedlings per treatment group, and data were analyzed using one-way ANOVA followed by Dunnett’s multiple-comparison test against the control group. Statistical significance was set at p < 0.05. We measured root length from 24 plants per treatment group, with measurements taken from 8 seedlings per replicate. Each treatment was replicated 3 times to ensure statistical reliability. All root samples were placed on top of an Epson Perfection V39 II Flatbed Portable Photo Scanner (Suwa, Japan). Excel, version 2019, Microsoft Corporation: Redmond, WA, USA, 2019, and the images were captured and saved for later processing. Root lengths of the individual root samples were calculated manually using ImageJ, version 1.53, National Institutes of Health: Bethesda, MD, USA, 2019 [48]. The value covered the length starting from the base of the root to the tip of the root. Statistical significance of the root length measurements was determined using R, version 4.5.1, R Foundation for Statistical Computing: Vienna, Austria, 2025, by comparing the plasma-treated samples with the control untreated samples.

4.4. Microbial Quantification Assay

The plasma treatments applied to the plants were like those previously described, including air and argon in a glow discharge plasma chamber, an atmospheric jet plasma handheld device, and a globe plasma, with durations of 10 s, 30 s, 1 min, and 5 min. Microbial counts were determined through serial dilution followed by plate counting. The resulting colony data were statistically analyzed using one-way ANOVA with Tukey’s post-hoc test for multiple comparisons. Seeds were set in square petri plates, stratified at 4 °C for 72 h, and transferred to a growth room with optimum growth parameters, including 12-h light and 12-h dark phases with temperature set at 21 °C and light intensity and humidity set respectively at 100 μmoL/m2/s and 45%. Seven seedlings per square plate were allowed to grow in each plate for about 2 weeks until they reached the two-leaf stage. The work was designed to include the quantification of the ACC deaminase-positive microbial strain O12 [49] and to estimate the growth of Arabidopsis plants exposed to varying plasma treatment conditions. An overnight culture of the O12 sample was inoculated to prepare a lawn. The OD of the culture was set at 0.001. The solution was mixed gently, and 10 mL of the culture was added onto each plate and kept for incubation for 2–3 min, followed by rinsing the solution and removing the culture from the plant surface. The inoculated plates were sealed and transferred to the growth room to be incubated for 72 h, followed by microbial quantification using serial dilution and then colony counting to estimate the number of colony-forming units [50].

4.5. Plasma Treatment and Parameters

The study utilized three plasma systems for sample treatment: a glow discharge plasma chamber, an atmospheric jet plasma handheld device, and a globe plasma (Figure 6). For each treatment, Arabidopsis seeds were placed in sterile petri dishes, standard 90 mm, Greiner Bio-One: Kremsmünster, Austria, 2019, and exposed to the plasma source at a fixed distance of 2 cm from the nozzle or plasma surface. In the glow discharge chamber, seeds were placed on a grounded electrode inside the vacuum chamber. For the atmospheric jet plasma, the handheld device was positioned vertically above the seeds. In the globe plasma setup, seeds were placed directly on the outer surface of the glass globe, and plasma filaments were allowed to contact the seed surface through the glass. For the glow discharge plasma chamber, two gas configurations were tested: air with a 45 cc/min flow rate and argon with a 35 cc/min flow rate. Both plasmas were initially formed below 0.35 Torr and subsequently maintained at 1.99 Torr of pressure. Each plasma condition was applied for four different durations: 10 s, 30 s, 1 min, and 5 min, designated as series A1–A4 for glow discharge air plasma-treated samples, B1–B4 for the atmospheric jet plasma-treated samples, C1–C4 for glow discharge argon plasma-treated samples, and D1–D4 for the globe plasma-treated samples. These varied exposure times allowed for the assessment of treatment duration effects on the experimental outcomes.

5. Conclusions

This research highlights plasma treatment as an effective and refined method for improving plant physiological traits, with the type of plasma and exposure time playing key roles in its success. Among the tested conditions, a 1-min globe plasma treatment consistently delivered the most favorable outcomes across various indicators of plant health. This study provides a comprehensive evaluation of plasma treatment parameters and their effects on plant performance, contributing valuable insights to the growing body of research on plasma-based agricultural innovations aimed at supporting sustainable farming and food security. Although optimal treatment conditions have been identified, further research is necessary to uncover the molecular mechanisms behind plasma-induced improvements, assess long-term impacts on plant growth and productivity, tailor protocols for different species and developmental stages, and explore the feasibility of large-scale implementation.

Author Contributions

Conceptualization, M.S.M., V.T., B.M., and C.K.; resources, B.M. and C.K.; writing—original draft preparation, B.M., C.K., and C.P.; writing—review and editing, K.M.P.-M., M.S.M., and V.T.; visualization, M.S.M., V.T., B.M., and C.K.; supervision, K.M.P.-M., M.S.M., and V.T.; project administration, K.M.P.-M., M.S.M., and V.T.; funding acquisition, M.S.M. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by NSF award IOS-2038872 to K.M.P.-M. and M.S.M., 2418230 to M.S.M. and NSF EPSCoR program for the funding and support through the RII-Track-1 Cooperative Agreement OIA-2148653 to V.T. and C.K.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding authors.

Acknowledgments

We acknowledge Jinbao Liu and Doni Thingujam for their support.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Chlorophyll estimation for plant plasma-treated samples. This figure shows the weight/weight (w/w) of Chl a per gram for different plasma treatments, including air and argon in a glow discharge plasma chamber, an atmospheric jet plasma handheld device, and a globe plasma, applied for durations of (a) 10 s, (b) 30 s, (c) 1 min, and (d) 5 min. * denote statistically significant differences compared to the control.
Figure 1. Chlorophyll estimation for plant plasma-treated samples. This figure shows the weight/weight (w/w) of Chl a per gram for different plasma treatments, including air and argon in a glow discharge plasma chamber, an atmospheric jet plasma handheld device, and a globe plasma, applied for durations of (a) 10 s, (b) 30 s, (c) 1 min, and (d) 5 min. * denote statistically significant differences compared to the control.
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Figure 2. Root length estimation for plasma-treated plant samples. This figure shows the primary root length (mm) for different plasma treatments, including air and argon in a glow discharge plasma chamber, an atmospheric jet plasma handheld device, and a globe plasma, applied for durations of 10 s, 30 s, 1 min, and 5 min. * denote statistically significant differences compared to the control.
Figure 2. Root length estimation for plasma-treated plant samples. This figure shows the primary root length (mm) for different plasma treatments, including air and argon in a glow discharge plasma chamber, an atmospheric jet plasma handheld device, and a globe plasma, applied for durations of 10 s, 30 s, 1 min, and 5 min. * denote statistically significant differences compared to the control.
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Figure 3. Root images for significant plasma treatments. This figure provides a visual comparison of root sizes for significant plasma treatments—(a–c)—against the control sample, (d).
Figure 3. Root images for significant plasma treatments. This figure provides a visual comparison of root sizes for significant plasma treatments—(a–c)—against the control sample, (d).
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Figure 4. Microbial quantification for plant plasma-treated samples. This figure shows the microbial counts for different plasma treatments, including air and argon in a glow discharge plasma chamber, an atmospheric jet plasma handheld device, and a globe plasma, applied for durations of (a) 10 s, (b) 30 s, (c) 1 min, and (d) 5 min. * denote statistically significant differences compared to the control.
Figure 4. Microbial quantification for plant plasma-treated samples. This figure shows the microbial counts for different plasma treatments, including air and argon in a glow discharge plasma chamber, an atmospheric jet plasma handheld device, and a globe plasma, applied for durations of (a) 10 s, (b) 30 s, (c) 1 min, and (d) 5 min. * denote statistically significant differences compared to the control.
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Figure 5. Chlorophyll estimation for plant plasma-treated samples. This figure shows the microbial counts for different plasma treatments, including air and argon in a glow discharge plasma chamber, an atmospheric jet plasma handheld device, and a globe plasma, applied for durations of (a) 10 s, (b) 30 s, (c) 1 min, and (d) 5 min. * denote statistically significant differences compared to the control.
Figure 5. Chlorophyll estimation for plant plasma-treated samples. This figure shows the microbial counts for different plasma treatments, including air and argon in a glow discharge plasma chamber, an atmospheric jet plasma handheld device, and a globe plasma, applied for durations of (a) 10 s, (b) 30 s, (c) 1 min, and (d) 5 min. * denote statistically significant differences compared to the control.
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Figure 6. Schematic representation of plasma systems used in the seed treatment. Arabidopsis seeds were treated with an atmospheric pressure plasma system, a glow plasma system, and a globe plasma system.
Figure 6. Schematic representation of plasma systems used in the seed treatment. Arabidopsis seeds were treated with an atmospheric pressure plasma system, a glow plasma system, and a globe plasma system.
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Mohan, B.; Karthik, C.; Pushpangathan, C.; Pajerowska-Mukhtar, K.M.; Thomas, V.; Mukhtar, M.S. Comparative Analysis of Plasma Technologies for Plant Growth Enhancement and Microbial Control: A Systematic Optimization Study. Int. J. Plant Biol. 2025, 16, 104. https://doi.org/10.3390/ijpb16030104

AMA Style

Mohan B, Karthik C, Pushpangathan C, Pajerowska-Mukhtar KM, Thomas V, Mukhtar MS. Comparative Analysis of Plasma Technologies for Plant Growth Enhancement and Microbial Control: A Systematic Optimization Study. International Journal of Plant Biology. 2025; 16(3):104. https://doi.org/10.3390/ijpb16030104

Chicago/Turabian Style

Mohan, Binoop, Chandrima Karthik, Chippy Pushpangathan, Karolina M. Pajerowska-Mukhtar, Vinoy Thomas, and M Shahid Mukhtar. 2025. "Comparative Analysis of Plasma Technologies for Plant Growth Enhancement and Microbial Control: A Systematic Optimization Study" International Journal of Plant Biology 16, no. 3: 104. https://doi.org/10.3390/ijpb16030104

APA Style

Mohan, B., Karthik, C., Pushpangathan, C., Pajerowska-Mukhtar, K. M., Thomas, V., & Mukhtar, M. S. (2025). Comparative Analysis of Plasma Technologies for Plant Growth Enhancement and Microbial Control: A Systematic Optimization Study. International Journal of Plant Biology, 16(3), 104. https://doi.org/10.3390/ijpb16030104

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