Large volume air plasma characteristics
The atmospheric pressure plasma formation was operated at 2000 Hz and 15 mm gap, as a function of increasing the input energy to the pulsed power supply system. As the input energy increases the plasma light emission intensity increase due to the increase in discharge current and applied voltage as shown in Figure 2. Small glows are formed on the pin tips at the initiation of the glow discharge when the lower applied voltage is established between the two electrodes. The glow discharges are generated at the outer pins initially and its luminousness is intense and spread to cover the discharge gape laterally and axially with increasing the applied voltage as shown in figure 2A-D. As well as the plasma gets diffuse and individual plasma columns merge with increasing the applied frequency and applied voltage [34]. The merging between individual plasma columns has been reported previously for a micro-hollow cathode sustained direct current air glow discharge [35].
Figure 3 shows a typical current-voltage waveform for the large volume atmospheric pressure air plasma that operated at 2000 Hz applied frequency, 15 mm discharge gap, 13 A discharge current, and 9.35 kV applied voltage. 100 ns was the estimated pulse duration of the positive half of the applied voltage waveform. The applied voltage and the discharge current measured the difference between the first (negative) and first (positive) peaks of the voltage and current waveforms. The discharge current increases with increasing the applied voltage, as the applied voltage increased from 7.09 kV to 9.52 kV, the discharge current increased from 3.86 A to 21.6 A. An arc discharge is formed when a further increase in the applied voltage is established. The discharge current variation with increasing the applied voltage indicates that the plasma is operating in the abnormal glow mode and resembling the point-to-plane negative corona discharges [36]. The operation of the large volume discharge plasma was limited below the glow to arc transition which depends on discharge gap distance and applied voltage and frequency.
The optical emission spectra are an indication of the plasma contents. The measured emission spectra from large volume atmospheric pressure plasma, at operating conditions of 2000 Hz and 1.5 cm discharge gap 2000 Hz, 13 A, 9.35 kV, indicated the presence of nitrogen molecule bands. The spectra show the presence of the first negative and second positive systems of the nitrogen molecule and the second positive system emission intensity was the highest in the investigated range between 200 nm and 500 nm (Figure 4). The emission spectra from NO, OH, and O radicals disappeared. The intensity of most of the detected nitrogen bands emission spectra increases with increasing discharge applied voltage.
The large volume atmospheric pressure air plasma gas temperature was measured side-on at the middle of the 15 mm discharge gap using an optical fiber bundle at the center of one of the plasma columns. The gas temperature was measured by evaluating (0,0) transition of the N2 second positive system rotational band. The gas temperature was determined by the best match between the measured 0-0 transition of the second positive system of nitrogen spectrum (C3Πu→B3Π) and the simulated one. Figure 5 presents the results of the estimated gas temperature of the generated plasma which was operated at operating conditions of 2000 Hz and 1.5 cm discharge gap 2000 Hz, 13 A, 9.35 kV. The results show that the gas temperature was in the range of 310 ± 20 oK. The results show that the generated plasma is an air non-thermal plasma operated at atmospheric pressure [35, 36].
Seedborne fungi
Several investigators indicated that cold plasmas have several mechanisms of action and can inhibit different fungi contaminating food and agricultural products [37, 38]. Their action includes the intracellular DNA breakage and protein oxidation of the outer membrane of fungi [39].
In this research, forty-one fungal colonies related to 13 species were recovered from M. oleifera dry seeds, about one-third of which were related to Aspergillus species (Table 1). A. flavus and A. niger were the dominant according to the quantitative occurrences. Cladosporum herbarum and Penicillium chrysogenum each were recovered 5 times from the investigated seeds. Rhizopus spp. were isolated in 6 colonies /10 g seeds. Each of Alternaria humicola and Fusarium oxysporum was represented in 3 colonies. The other species emerged in one or two colonies. On soaking, only 4 colonies were missed. There was a steady reduction in the total count of emerged fungi to 11 colonies and 2 colony/10 g seeds on the treatment of the seed soaked in water with 3.9 and 5.8 A cold plasma, respectively. With the same doses, only 8 and one colony/10 g were recovered from H2O2 soaked seeds, respectively. The higher doses (≥13 A) induced complete elimination of seedborne fungi. Seeds of different plants are commonly associated on or within the seeds with fungal pathogens causing rotting, wilting, or damping off. These fungi may remain inactive mycelial hyphae or spores within the seeds and cause new infections and disease dissemination to a new location [40]. El-mohamedy et al. [41] stated that the poor germination and low establishment of M. oleifera seedlings were related to the susceptibility of seeds to the soil fungi. Mitra et al. [42] indicated that the cold plasma treatment inactivates the seedborne microbial population, and consequently avoiding health risks and economic loss related to microbial contamination.
Table 1. Effect of cold atmospheric plasma on seedborne fungi associated with Moringa oleifera (colony/10 g dry seeds).
|
Untreated seeds
|
Treatment
|
Dry seeds (control)
|
Water-soaked seeds
|
Seeds treated with 3.9 A
|
Seeds treated with 5.8 A
|
Seeds treated with 13.0 A
|
A*
|
B*
|
A
|
B
|
A
|
B
|
Asperigillis flavus
|
6
|
4
|
2
|
1
|
0
|
0
|
0
|
0
|
A. candidus
|
1
|
1
|
0
|
0
|
0
|
0
|
0
|
0
|
A. terreus
|
2
|
2
|
1
|
1
|
0
|
0
|
0
|
0
|
Asperigillis niger
|
6
|
5
|
2
|
2
|
1
|
1
|
0
|
0
|
Alternaria humicola
|
3
|
3
|
0
|
0
|
0
|
0
|
0
|
0
|
Chaetomium globosum
|
1
|
1
|
0
|
0
|
0
|
0
|
0
|
0
|
Fusarium oxysporum
|
3
|
3
|
2
|
2
|
0
|
0
|
0
|
0
|
F. solani
|
1
|
1
|
0
|
0
|
0
|
0
|
0
|
0
|
Rhizopus oryza
|
4
|
4
|
1
|
0
|
0
|
0
|
0
|
0
|
R. nigricans
|
2
|
2
|
0
|
0
|
0
|
0
|
0
|
0
|
Cladosporum herbarum
|
5
|
4
|
1
|
1
|
0
|
0
|
0
|
0
|
Macrophomina phaseolina
|
2
|
2
|
0
|
0
|
0
|
0
|
0
|
0
|
Penicillium chrysogeum
|
5
|
5
|
2
|
1
|
1
|
0
|
0
|
0
|
Total count
|
41
|
37
|
11
|
8
|
2
|
1
|
0
|
0
|
*A= Seeds soaked in water, B= seeds soaked in 1% H2O2
Seed germination
The low doses of cold plasma (3.9 and 5.8 A) induced an increase in the seed germination of M. oleiferea which was more obvious and significant in the case of H2O2 soaked seeds. On the other, seed treatment with higher doses (13.0 and 21.6 A) induced marked inhibition in seed germination which was more pronounced in seedlings that emerged from H2O2 soaked seeds reaching 72.6 and 38.4% in the case of treatment with 13.0 and 21.6 A, respectively (Table 2). The results recorded for the germination rate match with those recorded for germination. The maximum leaf area was estimated for the seedling developed from 3.9 A cold plasma in the case of H2O2 soaked seeds followed by seeds soaked in H2O2 free water (Figure 6). The higher doses (13.0 and 21.6 A) significantly reduced leaf area compared with the control, though the reduction was more obvious with H2O2 soaked seeds.
Table 2. Percentage germination and average leaf area of Moringa oleifera seeds treated with different doses of atmospheric pressure cold plasma (J) for 10 minutes.
Dose of cold plasma (A)
|
Water-soaked seed
|
1% H2O2 soaked seed
|
% Germination
|
Germination rate
|
Leaf area (cm2)
|
% Germination
|
Germination rate
|
Leaf area (cm2)
|
0 (Control)
|
81.3±3.2
|
8.0±0.5
|
1.126±0.092
|
81.3±3.2
|
8.0±0.7
|
1.120±0.082
|
3.9
|
86.4±2.9
|
9.6±0.5
|
1.237±0.083
|
83.5±2.7
|
9.3±0.6
|
1.175±0.091
|
5.8
|
93.1±3.0
|
8.8±0.4
|
1.529±0.081
|
90.8±3.0
|
8.5±0.6
|
1.398±0.075
|
13.0
|
72.6±2.8
|
6.8±0.6
|
0.742±0.88
|
77.4±2.8
|
7.5±0.5
|
0.825±0.068
|
21.6
|
38.4±2.9
|
5.5±0.4
|
0.604±0.91
|
43.8±2.2
|
6.0±0.6
|
0.732±0.078
|
Although several investigations were carried out on the effects of plasma on seeds, however, the motivating activity of cold plasma as a mediator in the enhancement of seeds germination and seedling growth is not fully clear. It is thought that the facility of water uptake and the changes occurring on the external seed surface enhance the hydrophilic ability of seed and increase mobilization and solubilization of the reserve food in the seeds and interactions of cellular components, so initiating the growth and development of the seedlings [43]. The application of cold plasma may alter the seed surface wettability leading to more water absorption [44].
The reactive species emitted from cold plasma may cause breaking the seed dormancy [45] or induce scratching, clefts, or erosion in seed coat due to interaction with seed surface, so facilitating water uptake [18]. Dawood [46] indicated that treatment of M. oleifera seeds by cold plasma (RF-Ar low-pressure plasma) for one and five minutes improved the germination parameters as well as root and shoot potentials. The addition of H2O2 to the soaking water increases the efficiency of the cold plasma through the increased liberated active species that may contribute to plant increment in growth and development. Hydrogen peroxide molecule initiates the production of diverse other reactive oxygen radicals, such as superoxide, hydroxyl, and NOx, in cells. H2O2 primarily acts as a vital signaling molecule and stimulates the production of other signaling molecules such as enzymes, hormones, jasmonic acid, abscisic acid, and ethylene [47].
According to the literature review, the application of the cold plasma technique does not cause any change or mutation in the genetic material of seeds [48]. However, Šerá et al. [49] indicated that the active species of cold plasmas could pierce through the seed coats and excite natural signals such as growth factors [50]. This situation may induce regulation of the demethylation levels of certain genes [51], which leads to promotion in germination and seedling growth. Sidik et al. [52] working with corn plants showed that the seeds that were treated with 3 minutes of cold plasma treatment germinate faster and show a better growth rate related to the control seed. This revealed that cold plasma treatment is a suitable and standard technique to enhance seed germination and promote seedling growth of the plant.
Chlorophyll Content
The chlorophyll pigment contents of the seedlings grown from cold plasma-treated seeds differ significantly according to whether the seeds were soaked in water or 1% H2O2 solution. In lower plasma doses (3.9 and 5.8 A) there was a significant increase in chlorophyll content (chl a and chl b) of the seedlings that emerged from H2O2 soaked seeds rather than that free from H2O2. At higher doses of 13.0 and 21.6 A, the plasma was harmful, particularly when applied to H2O2 soaked water, where the chlorophyll a content of the seedling measured 6.1±0.4 and 5.2±0.4 mg/100 g for seedling developed from seed soaked in 1% H2O2 compared to 7.3 and 6.3 mg/100 g for seedlings appeared from seeds soaked in H2O2-free water, respectively (Table 3).
Table 3. Effect of atmospheric pressure cold plasma (A) applied for 10 minutes to M. oleifera dry or soaked seeds on chlorophyll content of seedling after 30 days emerged from water soaked or H2O2 soaked seeds
Dose of cold plasma (A)
|
Chlorophyll content (mg/100 g seedling leaf materials)
|
Water-soaked seeds
|
H2O2 soaked seeds
|
Chl a
|
Chl b
|
Chl a
|
Chl b
|
0 (Control)
|
6.7±0.3
|
5.3±0.3
|
6.9±0.4
|
5.4±0.5
|
3.9
|
7.3±0.4
|
6.2±0.5
|
7.6±0.4
|
6.4±0.4
|
5.8
|
7.9±0.4
|
6.6±o.4
|
8.6±0.3
|
6.9±0.4
|
13.0
|
7.3±0.5
|
5.6±0.4
|
6.1±0.4
|
4.4±0.3
|
21.6
|
6.3±0.3
|
4.3±0.5
|
5.2±0.4
|
3.7±0.5
|
High levels of chlorophyll in plasma-treated seeds can be attributed to increased physiological activity and photosynthesis in plants. Saberi et al. [53] reported that plasma treatment of winter wheat (Pishgam cultivar) for 180 seconds improved photosynthesis rate, chlorophyll content, and stomatal conductance by 34, 32, and 93%, respectively, compared with the control. However, other researchers have emphasized the positive effects of plasma on increasing chlorophyll content in tomato and maize [54, 55]. Šerá et al. [49] reported no significant changes in chlorophyll content in plasma treatment on rape seedlings. Jiafeng et al. [48] in their field experiments with wheat seeds treated with 80 W cold plasma, indicated that the chlorophyll content increased by 9.8% higher than those of the control, indicating that cold plasma treatment could promote the growth and the yield of treated plants.
Antioxidant activities
The antioxidant activity of M. oleifera seeds was evaluated in 30-day developed seedling after exposure of the seeds to varying doses of large volume plasma for 10 minutes. The lower doses (3.9 and 5.8 A) are simulative for the antioxidant activity of seedlings, particularly those that emerged from plasma-treated H2O2 pretreated seeds reaching 18.8 % compared to 11.5% in the case of the corresponding untreated seeds. The same trend was observed in the case of assessing total polyphenols, total flavonoids, ascorbic acid, and carotenoids reaching 320, 850, 1100, and 2460 µg/1g seed compared to 228, 710, 750, and 1440 µg/1g for the seedlings that emerged from untreated seeds under the same conditions, respectively (Figure 7). Ling et al. [56] indicated that the treatment of oilseed rape (Brassica napus L.) with cold plasma evidently increased the level of superoxide dismutase and catalase activities by 13.00-17.71% and 13.21-16.52%, respectively. Moreover, cold plasma treatment significantly induced an increase in the soluble sugar and protein contents suggesting that cold plasma treatment improve the drought resistance of the plant through the improvement of antioxidant enzyme activities, increasing osmotic-adjustment products, and reducing lipid peroxidation.
It has been reported that the low doses of cold plasma enhance the diversity of metabolic and physiological activities including antioxidant capacity, while it causes oxidative stress in seeds at higher doses [57]. Under such stress, plants are well equipped with a fundamental antioxidant defense system comprising enzymatic antioxidants, as well as non-enzymatic antioxidants, such as ascorbate to resist oxidative stress [58]. These antioxidants are important for motivating physiological and developmental processes and resisting stresses [59].
The higher plasma dose (>21.6 A) induces inhibition in total antioxidant compared to smaller doses although the value is still around the control value. However, total polyphenols, total flavonoids, ascorbic acid, and carotenoids were markedly decreased recording 120, 310, 500 and 1030 µg/1g for H2O2 soaked seeds exposed to 13.0 A for 10 minutes compared to the values recorded for control. A higher dose (> 21.6 A) induced remarkable inhibition for seedlings that emerged from plasma-treated seeds and was more evident for seedlings developed from H2O2 soaked seeds. At higher doses, more reactive ionic species are liberated, and the overproduction of these species may lead to oxidative stress damage to DNA, proteins, and lipids [60].