applied
sciences
Article
Influence of Plasma Activated Water Generated in a Gliding Arc
Discharge Reactor on Germination of Beetroot and Carrot Seeds
Piotr Terebun 1 , Michał Kwiatkowski 1 , Karol Hensel 2 , Marek Kopacki 3, *
1
2
3
*
Citation: Terebun, P.; Kwiatkowski,
M.; Hensel, K.; Kopacki, M.; Pawłat, J.
Influence of Plasma Activated Water
Generated in a Gliding Arc Discharge
and Joanna Pawłat 1, *
Institute of Electrical Engineering and Electrotechnologies, Lublin University of Technology, Nadbystrzycka
38a, 20-618 Lublin, Poland; p.terebun@pollub.pl (P.T.); m.kwiatkowski@pollub.pl (M.K.)
Department of Environmental Physics, Comenius University, Mlynská dolina F2, 842 48 Bratislava, Slovakia;
hensel2@uniba.sk
Department of Plant Protection, University of Life Sciences in Lublin, Leszczyńskiego 7, 20-069 Lublin, Poland
Correspondence: marek.kopacki@up.lublin.pl (M.K.); j.pawlat@pollub.pl (J.P.)
Abstract: One of the new methods of protecting and supporting plant growth is the use of lowtemperature plasma. The aim of this study is to evaluate the feasibility of using plasma activated
water produced in an atmospheric pressure gliding arc reactor for germination of beetroot (Beta
vulgaris) and carrot (Daucus carota) seeds. The study was carried out for different plasma treatment
times of water (5, 10 and 20 min) and with fixed geometry and power of the discharge system, using
air as the working gas. The effect on germination was evaluated based on the fraction of germinated
seeds and their length at 7 and 14 days after treatment. Analysis of fungi present on the seed surface
and imaging of the seed surface using scanning electron microscopy (SEM) were auxiliary methods
to evaluate the type of treatment effect. In the case of beetroot, a positive effect on the number and
length of germinated seeds was observed, which increased with increasing treatment time. This
effect can be attributed, among other things, to the surface changes observed on microscopic photographs. In the case of carrot seeds, a more significant positive effect on germination was observed.
Fungal decontamination effect was relatively weaker than with the use of the chemical method with
sodium hypochlorite.
Reactor on Germination of Beetroot
and Carrot Seeds. Appl. Sci. 2021, 11,
6164. https://doi.org/10.3390/
Keywords: plasma activated water (PAW); gliding arc discharge (GAD); germination; carrot seed;
beetroot seed
app11136164
Academic Editor: Joachim Müller
1. Introduction
Received: 24 May 2021
Accepted: 25 June 2021
Published: 2 July 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
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Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Currently, one of the main tasks of plant protection is the implementation of innovative
and safe methods to reduce the occurrence of agrophages in crops [1,2]. This is related to
the implementation of the concept of sustainable agriculture, promoting the production
of high quality food in a socially responsible manner, rational use of natural resources
and a reduction in the use of chemical plant protection products [3–6]. Excessive and
careless use of pesticides contributes not only to resistant breeds of agrophages, but also to
environmental pollution and residues in the raw materials produced [7]. One alternative
method of pesticide-free crop protection may be plasma treatment. Plasma can affect
living cells through the action of active particles generated in the plasma (mainly reactive
oxygen and nitrogen species—RONS), the action of charged plasma particles, radiation
over a wide range of wavelengths, and shear stresses and drying [8–10]. The mechanism of
plasma action on harmful microorganisms is multistage and mainly involves permanent
damage of the cell wall, cytoplasmic membrane, and then intracellular structures, genetic
material and the enzyme apparatus [11–13]. Plasma can be used to decontaminate seed
and seedling material [14–22] and have positive effects on physiological processes in plants
and plant seedlings [23–32]. For example, the work of Jiang et al. indicated the effects
of low-temperature plasma on seed germination, seedling growth, root morphology, and
nutrient uptake in tomato [33]. Many researchers confirm the positive effect of plasma
Appl. Sci. 2021, 11, 6164. https://doi.org/10.3390/app11136164
https://www.mdpi.com/journal/applsci
i. 2021, 11, x FOR PEER REVIEW
2 of 16
be used to decontaminate seed and seedling material [14–22] and have positive effects on
2 of 15
processes in plants and plant seedlings [23–32]. For example, the work of
Jiang et al. indicated the effects of low-temperature plasma on seed germination, seedling
growth, root morphology, and nutrient uptake in tomato [33]. Many researchers confirm
the positive effect of plasma on the process of plant rooting especially on increasing the
on the process of plant rooting especially on increasing the length and number of roots
length and number of roots formed [34,35].
formed [34,35].
One method of using plasma treatment may be to use plasma activated water (PAW),
One method of using plasma treatment may be to use plasma activated water (PAW),
which may contain
active
(such particles
as dissolved
ozone,
hydrogen
peroxide,
nitrates,
which
may particles
contain active
(such
as dissolved
ozone,
hydrogen
peroxide, nitrates,
nitrites, peroxynitrites,
OH
radicals,
etc.)
produced
directly
in
the
discharge
or
their
nitrites, peroxynitrites, OH radicals, etc.) produced directly in theby
discharge
or by their
reaction with water,
depending
on depending
the type of on
discharge
the working
gas
[36]. gas used [36].
reaction
with water,
the typeand
of discharge
and
theused
working
Then, PAW canThen,
be used
to can
remove
harmful
organisms
[37–45],
either [37–45],
directly either
on growing
PAW
be used
to remove
harmful
organisms
directly on growing
plants [46,47] or
by
aiding
in
the
germination
process
[48,49].
From
the
perspective
ofperspective of
plants [46,47] or by aiding in the germination process [48,49]. From the
stability over time,
hydrogen
peroxide
and lower
pH and
in PAW
stability
over time,
hydrogen
peroxide
lowerare
pHprobably
in PAW the
are most
probably the most
influential factors
in
fungicidal
activity.
The
treatment
effect
depends
on
both
the the discharge
influential factors in fungicidal activity. The treatment effect depends on both
discharge parameters
and
the
type
of
plant
being
treated.
The
purpose
of
this
study
is
to is to evaluate
parameters and the type of plant being treated. The purpose of this study
evaluate the feasibility
of
using
PAW
generated
by
a
gliding
arc
discharge
(GAD)
to
aid
the feasibility of using PAW generated by a gliding arc discharge (GAD) to aid in the
in the germination
of carrotof
and
beetroot
In seeds.
additionIntoaddition
the results
on the
fraction
germination
carrot
and seeds.
beetroot
to the
results
on the fraction of
of germinated germinated
seeds and the
length
sprouts,
obtained
from
surface
imaging
and
seeds
andofthe
length those
of sprouts,
those
obtained
from
surface
imaging and the
the analysis of analysis
the type and
amount
of fungi
present
on the
seeds, on
conducted
viaconducted
scanning via scanning
of the
type and
amount
of fungi
present
the seeds,
electron microscopy
(SEM),
were also
usedwere
to evaluate
thetoeffect
of plasma.
Theofresults
electron
microscopy
(SEM),
also used
evaluate
the effect
plasma. The results
were comparedwere
withcompared
one of thewith
classic
methods
of
plant
decontamination—treatment
with
one of the classic methods of plant decontamination—treatment
with
sodium hypochlorite
(NaOCl).
sodium hypochlorite (NaOCl).
Appl. Sci. 2021, 11, 6164
physiological
2. Materials
2. Materials and
Methods and Methods
2.1. Treated Seeds
2.1. Treated Seeds
Edible
seeds of F1
thevariety
AFALON
F1 variety
Moravo Seed Ożarów
Edible carrot seeds
of carrot
the AFALON
(Daucus
carota,(Daucus
Moravocarota,
Seed Ożarów
Mazowiecki,
Poland)
and
beetroot
seeds
of
the
CYLINDRA
variety
(Beta vulgaris, W.
Mazowiecki, Poland) and beetroot seeds of the CYLINDRA variety (Beta vulgaris, W.
1a).
For
Legutko,
Jutrosin,
Poland)
were
used
in
the
research
(Figure
Legutko, Jutrosin, Poland) were used in the research (Figure 1a). For each treatmenteach treatment
condition,
10 seeds were
randomly
selected,
and the measurements
repeated 5 times.
condition, 10 seeds
were randomly
selected,
and the
measurements
were repeated 5were
times.
Figure 1. (a)Figure
Photographs
of treated seeds,
(b) schematic
the experiment.
1. (a) Photographs
of treated
seeds, (b)ofschematic
of the experiment.
2.2. Plasma Activated
WaterActivated Water
2.2. Plasma
Plasma was generated
in generated
a single-phase
gliding arcgliding
reactorarc
(GAD)
operating
at
Plasma was
in a single-phase
reactor
(GAD) operating
at atmoatmospheric pressure;
this
process
has
been
described
in
greater
detail
in
previous
works
spheric pressure; this process has been described in greater detail in previous works [50,51].
[50,51]. The discharge
system system
consisted
of two copper
2 mm thick
andthick
80 mm
The discharge
consisted
of two electrodes,
copper electrodes,
2 mm
and 80 mm long,
with an angle of 12◦ between them, placed in a glass tube with an inner diameter of
50 mm. The working gas (air) was blown out through the glass nozzle near the ignition
area with a flow rate of 7.33 dm3 /min, adjustable with glass tube variable area flow meter
(Zakłady Automatyki “ROTAMETR”, Gliwice, Poland). The reactor was powered by an
Appl. Sci. 2021, 11, 6164
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RMS (root-mean-square) voltage of 680 V (3.8 kV peak voltage), frequency of 50 Hz and
apparent power of 40 VA. To obtain plasma activated water, 20 mL of distilled water, at
22.6 ◦ C, was placed in a glass vessel with a diameter of 60 mm and with a distance of
20 mm between the electrodes and the water surface (Figure 1b).
The water temperature after plasma treatment of 5, 10 and 20 min, measured with
a K-type thermocouple connected to the DT-847U meter (Yu Ching Technology, Taipei,
Taiwan), was 27 ◦ C, 29.1 ◦ C and 30.7 ◦ C, respectively.
The colorimetric method using TiOSO4 (Titanium(IV) oxysulfate) solution (Merck,
Darmstadt, Germany) and colorimetric Griess assay (Cayman Chemicals, Ann Arbor,
USA) were used for the measurement of hydrogen peroxide (H2 O2 ) and nitrites (NO2 − ),
respectively. The obtained concentrations of active species increased with the plasma
treatment time and are summarized in Table 1. The GAD plasma source is able to generate
high concentrations of nitrogen reactive species but H2 O2 concentration was low, which is
in a good accordance with previous data [50].
Table 1. Concentration of selected RONS.
Plasma Treatment
Time [min]
H2 O2
[µM]
NO2 −
[mM]
pH
5
10
20
6±1
7±3
12 ± 5
1.9 ± 0.4
2.4 ± 0.3
2.9 ± 0.6
4.2 ± 0.2
3.7 ± 0.1
3.3 ± 0.3
2.3. Germination Rate
Immediately after the plasma treatment of water, the experiment with seeds was
carried out in three parts: first, the seeds were treated with PAW, e.g., water was poured
over 10 seeds of each species placed in glass flasks and left to soak for one hour. Then,
seeds were transferred to the Petri dishes with the mineral nutrient medium solution (the
composition of the solution is shown in Table 2). In the last step, mycological analysis
was performed.
Table 2. Composition of mineral medium.
Substance
Quantity
Saccharose
Agar
NH4 NO3
MgSO4 × 7 H2 O
NH2 PO4
FeCl3 × 7 H2 O
ZnSO4 × 7 H2 O
CuSO4 × 7 H2 O
MnSO4 × 7 H2 O
38 g
20 g
0.7 g
0.3 g
0.3 g
trace amounts
trace amounts
trace amounts
trace amounts
In order to compare plasma treatment with traditional decontamination methods, a
series of measurements was performed using a sodium hypochlorite. For this purpose,
10 seeds were sterilized in 10% NaOCl solution for 60 s and then washed 3 times for 3 min
in distilled water. NaOCl (POCH Odczynniki Chemiczne, Avantor, Poland) was diluted
to proper concentration on-site immediately before application. Seed samples from the
same batch were used for the tests. Then, the seeds were placed on the plates, similarly
to the plasma treatment and control. All seeds were in sterile growth mineral medium,
in the cultivation chamber with controlled atmosphere, under the following conditions:
20–22 ◦ C without light with 65% humidity. The amount of germinated beet and carrot seeds
Appl. Sci. 2021, 11, 6164
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was measured after 7 days for germination energy GEN and after 14 days for germination
capacity GC [52]. Both coefficients were then calculated from the following equation:
G=
n
· 100%
nT
(1)
where: n—the number of seeds germinated at time t; nT —the total number of sown seeds.
2.4. Fungi on the Seed Surface
For additional series with the same treatment conditions, in the germination study
a mycological analysis was performed using the method of artificial cultures [53]. The
medium with the composition presented in Table 1 was supplemented with distilled water
to the volume of 1000 mL, and then sterilized for 20 min in an autoclave, at a temperature
of 121 ◦ C and a pressure of 1 atmosphere. The dishes with the seeds lined up were placed
at 20–22 ◦ C for 7 days without light. After one week, the plates were inspected, and the
grown fungus colonies were cleaved on slants with potato-glucose agar (PDA). Then,
segregation was performed on the basis of microscopic and macroscopic features. The
obtained fungal isolates were sorted and labeled according to species using studies, keys
and monographs [54–57].
2.5. Surface Imaging Using an Optical Microscope and SEM
Morphology of the seeds was evaluated using a KEYENCE VHX-5000 digital microscope (Osaka, Japan). Images of the samples were taken immediately after one hour of the
seeds’ imbibition in plasma activated water.
For more detailed visualization of the surface of Beta vulgaris seeds after PAW treatment,
the treated samples were naturally dried for one hour and applied to the mounting surface
(carbon disc). A layer of gold (~15 nm) was then sputtered onto the samples for imaging
using a Phenom SEM microscope (Thermo Fisher Scientific, USA) at a voltage of 10 kV.
2.6. Statistical Analysis
StatSoft’s Statistica 8.0 software was used for the analysis of the experimental data.
Statistical differences between groups were examined with use of two-way analysis of
variance (ANOVA). Tukey’s test was used to analyze the significance of differences between
mean values (α ≤ 0.05). For the data obtained with measurement of the same objects at
different times, the results are correlated due to the use of the ANOVA Repeated Measures
Analysis of Variance.
3. Results and Discussion
3.1. Germination Rate
Beta vulgaris germination results for control, NaOCl and PAW (using 5, 10 and
20 min of GAD treatment of water as PAW5′ , PAW10′ and PAW 20′ ) are summarized
in Figure 2. Compared to the control, which had a rather high variability within samples,
PAW treatment allowed for an increase in the average value of both GEN and GC . The
difference in germination energy was evident for PAW after 5 min of plasma treatment, for
which the fraction of germinated seeds increased by 16%, performing better than sodium
hypochlorite treatment. Similar GEN results were obtained for a time of 10 min plasma
treatment of water; however, this treatment time also allowed for an increase in GC , as
opposed to PAW5’, which had a shorter treatment time that accelerated initial growth
followed by no germination of the remaining seeds. The best results were obtained for
PAW with a treatment time of 20 min, for which all seeds from all samples germinated after
7 days. In the case of beetroot seeds, the highest ratio was obtained with the application of
water treated with plasma for 20 min; this was also the case for carrot seeds in combination
with sodium hypochlorite.
Appl. Sci. 2021, 11, x FOR PEER REVIEW
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Appl. Sci. 2021, 11, 6164
5 of 16
5 of 16
after 7 days. In the case of beetroot seeds, the highest ratio was obtained with the
after 7 days. In the case of beetroot seeds, the highest ratio was obtained with the
application of water treated with plasma for 20 min; this was also the case for carrot seeds
5 of 15
application of water treated with plasma for 20 min; this was also the case for carrot seeds
in combination with sodium hypochlorite.
in combination with sodium hypochlorite.
Figure
2. Germination
values and
standard deviation).
Figure
Germinationofof
ofBeta
Betavulgaris
vulgaris(mean
(mean
Figure 2.
2. Germination
Beta
vulgaris
(mean values
values and
and standard
standard deviation).
deviation).
The
germination results
obtained for
Daucus carota
are shown
in Figure
3.
The
The germination
germination results
results obtained
obtained for
for Daucus
Daucus carota
carota are
are shown
shown in
in Figure
Figure 3.
3.
Figure3.3.Germination
GerminationofofDaucus
Daucuscarota
carota(mean
(meanvalues
valuesand
andstandard
standarddeviation).
deviation).
Figure
Figure 3. Germination of Daucus carota (mean values and standard deviation).
Forthe
thetested
testedseeds,
seeds,the
thegermination
germinationof
ofthe
thecontrol
controlsamples
sampleswas
waslow
lowand
and did
did not
not
For
For10%
theeven
tested
seeds,
the germination
oftreatment
the control
samples
wasanlow
and did
not
exceed
after
14
days.
As
the
plasma
time
increased,
increase
in
G
EN
exceed 10% even after 14 days. As the plasma treatment time increased, an increase in GEN
exceed
10%
even
after
14
days.
As
the
plasma
treatment
time
increased,
an
increase
in
G
EN
was
evident,
which
more
than
doubled
for
a
time
of
20
min.
However,
the
best
results
at
was evident, which more than doubled for a time of 20 min. However, the best results at
wasend
evident,
more
than
doubled
for for
a time
of which
20 min.undergone
However, plasma
the besttreatment
results at
the
ofthe
thewhich
test(14
(14
days)
were
obtained
PAW,
the
end of
test
days)
were
obtained
for PAW,
which undergone
plasma treatment
the shorter
end of the
test
(14
days)
were
obtained
for PAW,
which
undergone
plasma
treatment
for
times
(5
and
10
min),
where
the
average
G
value
was
almost
three
times
higher
for shorter times (5 and 10 min), where the average GCCvalue was almost three times higher
for
shorter
times
(5
and
10
min),
where
the
average
G
C
value
was
almost
three
times
higher
thanthe
thecontrol
controlsample.
sample.For
Forthe
thetested
testedseeds,
seeds,the
thebest
bestresults
resultswere
wereobtained
obtainedfor
forthe
thesodium
sodium
than
than the control
sample. For
the tested
seeds, the
best results
obtainedsamples
for the sodium
hypochlorite
treatment,
although
the results
obtained
from were
the different
varied
hypochlorite treatment, although the results obtained from the different samples varied
hypochlorite
treatment, although the results obtained from the different samples varied
quite
significantly.
quite significantly.
quite significantly.
3.2. Sprout Length
In the case of the total length of sprouts, the highest results were obtained in both
plant species for seeds treated with sodium hypochlorite.
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Appl. Sci. 2021, 11, 6164
3.2. Sprout Length
6 of 16
6 of 15
In the case of the total length of sprouts, the highest results were obtained in both
plant species for seeds treated with sodium hypochlorite.
The
experimental condition
condition for
for Beta
Beta vulgaris
vulgaris are
Theaverage
average sprout
sprout lengths
lengths from
from each
each experimental
are
shown
in
Figure
4.
shown in Figure 4.
Figure4.4.Sprout
Sproutlength
lengthofofBeta
Betavulgaris
vulgaris(mean
(meanvalues
valuesand
andstandard
standarddeviation).
deviation).
Figure
Whencomparing
comparingmean
meansprout
sproutlengths,
lengths,after
after77 days,
days, each
each treatment
treatment type
type allowed
allowed
When
greater
lengths,
with
a
slightly
greater
advantage
for
PAW
with
20
min
plasma
treatment
greater lengths, with a slightly greater advantage for PAW with 20 min plasma treatment
46%increase
increaseover
overthe
thecontrol).
control).After
After14
14days,
days,the
thedifferences
differencesfor
forthe
theplasma
plasmatreatment
treatment
(a(a46%
are
less
noticeable
(4
to
17%
increase
over
the
control),
with
a
large
increase
in
length
for
are less noticeable (4 to 17% increase over the control), with a large increase in length for
the
sodium
hypochlorite
treatment
(32%
increase
with
a
relatively
large
difference
between
the sodium hypochlorite treatment (32% increase with a relatively large difference
the individual
samples).samples).
As for the
at the 7th day
of observation,
between
the individual
Assprouts’
for theproduction
sprouts’ production
at the
7th day of
application
of
PAW5’
produced
a
visible
improvement
in
the
germination
process.
With an
observation, application of PAW5' produced a visible improvement in the germination
extension in the observation time, the stimulation effect became weaker, and for 14 days
Appl. Sci. 2021, 11, x FOR PEER REVIEW
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process. With an extension in the observation time, the stimulation effect became weaker,
was clearly visible only for 20 min-treated PAW and NaOCl.
and for 14 days was clearly visible only for 20 min-treated PAW and NaOCl.
The results obtained for Daucus carota are shown in Figure 5.
The results obtained for Daucus carota are shown in Figure 5.
Figure
Figure 5.
5. Sprout
Sprout length
length of
of Daucus
Daucus carota
carota (mean
(mean values
values and
and standard
standard deviation).
deviation).
These seeds react in a slightly different manner than Beta vulgaris as a very weak
stimulative effect can be observed after 7 days of treatment; the best results were obtained
for sodium hypochlorite (250% increase), while PAW treatment was again the most
effective, with the longest water treatment time with plasma (40% increase compared to
the control). Such a PAW still had relatively low concentration of H2O2, with
Appl. Sci. 2021, 11, 6164
7 of 15
These seeds react in a slightly different manner than Beta vulgaris as a very weak
stimulative effect can be observed after 7 days of treatment; the best results were obtained
for sodium hypochlorite (250% increase), while PAW treatment was again the most effective, with the longest water treatment time with plasma (40% increase compared to the
control). Such a PAW still had relatively low concentration of H2 O2 , with decontaminative properties and a reasonably high concentration of nitrites with known antimicrobial
properties, especially at the low pH, which together boosted plant’s growth and inhibited
the development of certain fungal colonies. Nitrites are known for their fertilizing properties and support further plant development. After 14 days, a very significant increase
in sprout length was noticeable for all types of treatment. For NaOCl, this was more than
a thirteenfold increase over the control. For PAW treatment, the best effect was obtained
for the shortest time of PAW treatment with plasma (650% increase), followed by 20 and
10 min of treatment (505% and 300% increase, respectively), which could be explained by
the attaining of a kind of balance between RONS and pH, with the location of the above
factors in a plant environmental tolerance zone. On the other hand, the germination boost
that was clearly visible for NaOCl treatment could be explained by a strong correlation
between the germination process and fungal decontamination, which is described in the
next chapter.
3.3. Statistical Analysis
Results of statistical analysis are depicted in Tables 3 and 4. The best results, with a
statistically significant change in the case of Beta vulgaris, were obtained for PAW, with a
treatment time of 20 min, for which all seeds, from all samples, germinated after 7 days.
For the tested Daucus carota seeds, the best results, with a statistically significant change,
were obtained for the sodium hypochlorite treatment, although the results obtained from
the different samples varied quite significantly.
Table 3. Statistical analysis of germination results. The letter indicators next to the means determine
the statistically homogeneous groups.
Beta Vulgaris
Daucus Carota
GEN
GC
GEN
GC
Control
0.78 b
0.9 ab
0.08 abcd
0.08 abcd
NaOCl
0.9 ab
0.94 ab
0.4 ef
0.42 f
5′
0.94 ab
0.94 ab
0.06 ab
0.22 cdef
PAW
10′
0.94 ab
0.98 a
0.1 ac
0.22 bdef
PAW
20′
1.0 a
1.0 a
0.18 abcde
0.18 abcde
PAW
3.4. Fungi on the Seed Surface
Fungal infestations constitute a persistent problem for improperly stored seeds, which
brings losses of the seeding material, generates additional costs and is inappropriate from
a sustainable ecology point of view. During our observation of basic microbiota present
in the examined seeds, certain trends were observed when assessing the infestation of
seeds of selected species by fungi. The seeds were highly contaminated. With longer GAD
application times to water, a reduction in the number of fungi inhabiting the seeds can be
observed. A total of 13 fungal species were identified in Beta vulgaris. Colony counts for
each species and treatment condition are summarized in Table 5.
′′′ ′
′′′ ′
′′′ ′
Appl. Sci. 2021, 11, 6164
8 of 15
Table 4. Results of statistical analysis.
Beta Vulgaris
Daucus Carota
Germination rate
Sprouts’ length
Table 5. Fungal species on Beta vulgaris.
Species of Fungus
Control
NaOCl
GA5′
GA10′
Alternaria alternata (Fr.) Keissl.
Aspergillus niger Tiegh
Botrytis cinerea Pers.
Chaetomium cochliodes Palliser
Clonostachys rosea (Link) Schroers
Epicoccum nigrum Link
Fusarium solani (Mart.) Sacc.
Penicillium expansum Link
Penicillium nigricans K.M. Zal.
Truncatella truncata Lev.
Trichoderma harzianum Rifai
Trichoderma koningii Oudem.
Trichothecium roseum (Pers.) Link
Total
2
3
1
0
0
0
2
0
1
2
0
2
0
13
0
0
0
0
16
0
0
0
0
0
4
2
0
22
1
2
0
5
0
10
0
0
2
0
1
0
0
21
0
0
0
0
0
8
0
1
0
0
0
3
0
12
GA20′
11
0
0
2
0
0
0
2
0
0
′′′ ′ 1
0
1
17
Total
′′′ ′
14
5
1
7
16
18
2
3
3
2
′′′ 6′
7
1
85
In total, the largest numbers of colonies were observed for Epicoccum nigrum, Clonostachys
rosea and Alternaria alternata. Compared to the control, all PAW treatments eliminated fungi
such as Botrytis cinerea, Fusarium solani and Truncatella truncata with the same efficiency as the
sodium hypochlorite treatment. For Aspergillus niger, the reduction occurred only for PAW
Appl. Sci. 2021, 11, 6164
9 of 15
with a treatment time of 10 min. On the other hand, for some treatment times, more colonies
of Alternaria alternata, Epicoccum nigrum and Penicillium expansum were observed.
The results obtained for Daucus carota are summarized in Table 6.
Table 6. Fungal species on Daucus carota.
Species of Fungus
Control
NaOCl
GA5′
GA10′
GA20′
Total
Alternaria alternata (Fr.) Keissl.
Alternaria radicina Meier, Drechsler & E.D. Eddy
Aspergillus niger Tiegh
Chaetomium cochliodes Palliser
Cladosporium sp.
Clonostachys rosea (Link) Schroers
Epicoccum nigrum Link
Fusarium avenaceum (Fr.) Sacc.
Mucor mucedo Fresen.
Penicillium expansum Link
Stemphylium botryosum Wallr.
Trichoderma harzianum Rifai
Trichoderma koningii Oudem.
Total
49
7
2
0
0
0
0
0
0
0
0
2
0
60
0
4
6
0
15
2
1
0
0
0
12
2
0
42
48
0
2
1
0
0
0
0
0
0
0
0
0
51
48
0
0
0
0
0
0
0
2
0
0
0
1
51
48
0
2
0
0
0
0
2
0
3
0
0
0
55
193
11
12
1
15
2
1
2
2
3
12
4
1
259
The highest number of colonies was observed for the Alternaria alternata species,
which was completely resistant to PAW treatment but was entirely removed by NaOCl.
On the other hand, high numbers of colonies were observed for the species Cladosporium
sp. and Stemphylium botryosum, which were only detected for the treatment with sodium
hypochlorite. This may result from the surface effect of PAW, the relatively low concentration of disinfective hydrogen peroxide, and also from high primary contamination of
seeds, especially carrots, as a result of improper storage. PAW treatment resulted in a
complete reduction in Alternaria radicina and Trichoderma harzianum, which were present
in both control and NaOCl treated samples. It also caused a slight increase in Chaetomium
cochliodes, Fusarium avenaceum, Mucor mucedo, Penicillium expansum and Trichoderma koningii,
which usually occurred for longer processing times. In the case of the cheapest treatment
option for the feed gas (air), the RONS composition present in plasma activated water had
mild fungitoxic effects. Such a treatment strongly depended on the fungal species; thus,
positive point is a kind of treatment selectivity, which can be achieved using PAW. It has
to be pointed out that PAW reveals basic surface activity. However, in some cases, high
contamination, with the presence of pathogens that are located in the deeper zones of seeds,
also cannot be removed by traditional chemical treatment techniques such as NaOCl.
3.5. Surface Imaging Using an Optical Microscope and SEM
In order to emphasize the differences between two types of tested seeds and also
the differences visible in the obtained results, the seeds’ structures were described on
the basis of the literature data, and microscopic analyses were performed. Beta vulgaris
belongs to the complex Eudicots, with a perispermic seed structure. In this case, water and
oxygen penetration can be limited by thick, hard pericarp tissue surrounding the internal
botanical seed. Morphologically, an ovary cap with the remnants of the stigma covers
the pericarp’s upper part. On the opposite side of the seed, the basal pore is located in a
position that allows for water intake. Pericarp consists of three layers with different sizes
of crystalized chemical compounds, which tend to be bigger with depth. The first of two
dense internal sclerenchymal layers is formed of small, multilayer-wall sclereids, followed
by the second layer of thinner cell wall sclereids with crystal clusters inside, and the rather
loose parenchyma cells located externally. The analysis of water extract from the pericarp
revealed predominating cations such as potassium, sodium, magnesium, calcium, chlorine,
sulphur and anions with nitrate, phosphate, chloride and sulphate oxalate ions dominating,
pericarp’s upper part. On the opposite side of the seed, the basal pore is located in a
position that allows for water intake. Pericarp consists of three layers with different sizes
of crystalized chemical compounds, which tend to be bigger with depth. The first of two
dense internal sclerenchymal layers is formed of small, multilayer-wall sclereids, followed
by the second layer of thinner cell wall sclereids with crystal clusters inside, and the rather
Appl. Sci. 2021, 11, 6164
10 of 15
loose parenchyma cells located externally. The analysis of water extract from the pericarp
revealed predominating cations such as potassium, sodium, magnesium, calcium,
chlorine, sulphur and anions with nitrate, phosphate, chloride and sulphate oxalate ions
whichform
form
the
osmotic
potential
within
pericarp,
which
may
cause
delays in the seed
dominating, which
the
osmotic
potential
within
pericarp,
which
may
cause
delays
germination
process
[58–63].
in the seed germination
process
[58–63].
When
both
the opticaland
microscope
and electron
the scanning
electron microscope
When using both
theusing
optical
microscope
the scanning
microscope
(Figures
6
and
7),
imaged
samples
are
characterized
by
oval
shaped
parenchymal
cells,
(Figures 6 and 7), imaged samples are characterized by oval shaped parenchymal cells,
of
of
random
size,
with
a
very
developed,
concaved
structure.
The
cells
are
not
densely
random size, with a very developed, concaved structure. The cells are not densely packed
packed
and
spaces
observed.
After
PAW treatment,
and intercellular
spaces
canintercellular
be observed.
After can
the be
PAW
treatment,
thethe
structure
became the structure
became and
folded
wrinkled,
and
revealed
signs of rupture
in comparison to the
folded and wrinkled,
alsoand
revealed
signs
of also
rupture
in comparison
to the control
control
sample. Changes
arewith
noticeable
forplasma
PAW with
5 min of
plasma
treatment. On the
sample. Changes
are noticeable
for PAW
5 min of
treatment.
On
the other
other
hand,
besides
the
undulating
structure,
longer
times
resulted
in
the
flattening of the
hand, besides the undulating structure, longer times resulted in the flattening of the outer
outer
edges
of
the
cells.
edges of the cells.
i. 2021, 11, x FOR PEER REVIEW
11 of 16
′ ; (c) PAW 20′ .
Figure microscope
6. Optical microscope
images
of Beta
vulgaris
at 500× magnification.
Control(b)
(water);
Figure 6. Optical
images of Beta
vulgaris
at 500
× magnification.
(a) Control(a)(water);
PAW 5(b)
PAW 5′; (c) PAW 20′.
Figure
7. SEM
images
of Beta
vulgaris
at 1000×
magnification.
Control
(water);
PAW
5′; PAW
(c) 20′ .
Figure
7. SEM
images
of Beta
vulgaris
at 1000
× magnification.
(a) (a)
Control
(water);
(b) (b)
PAW
5′ ; (c)
PAW 20′.
Carrot seeds are relatively small and they belong to the schizocarpic fruits. Their
Carrot seeds
are relatively
small and
to the
fruits. Their
external
layer is formed
by athey
thinbelong
seed coat
andschizocarpic
it has overhanging
beards. Pericarp is
external layer is
formedjoined
by a thin
it hasofoverhanging
beards. Pericarp
is
partially
to theseed
seedcoat
andand
consists
the epicarp, mesocarp
and endocarp.
The bulk
partially joinedoftothe
theseed
seedincorporates
and consiststhick-walled
of the epicarp,
mesocarptissue
and endocarp.
The
endosperm
embodying
anbulk
embryo [64–66].
of the seed incorporates
thick-walled
endosperm
tissue embodying
embryoof[64–66].
As shown
in Figure
8, on the weavy-ribbed
top an
structure
the coat, the distinctive
As shownelongating
in Figure 8,beards
on theadjoined
weavy-ribbed
of in
thethe
coat,
the distinctive
to the top
ribsstructure
are visible
control
sample. PAW treatment
caused
visibletochanges
enhanced
the ribsPAW
and removal
of beards. Beard
elongating beards
adjoined
the ribssuch
are as
visible
in theswelling
control of
sample.
treatment
removalsuch
is also
a common
processofduring
processBeard
of the carrot seeds,
caused visible changes
as enhanced
swelling
the ribsthe
andscarification
removal of beards.
takes place
in during
order tothe
enhance
germination
to save
space
during the seed
removal is alsowhich
a common
process
scarification
processand
of the
carrot
seeds,
packaging
process.
which takes place
in order
to enhance germination and to save space during the seed
packaging process.
of the seed incorporates thick-walled endosperm tissue embodying an embryo [64–66].
As shown in Figure 8, on the weavy-ribbed top structure of the coat, the distinctive
elongating beards adjoined to the ribs are visible in the control sample. PAW treatment
caused visible changes such as enhanced swelling of the ribs and removal of beards. Beard
removal is also a common process during the scarification process of the carrot seeds,
Appl. Sci. 2021, 11, 6164
which takes place in order to enhance germination and to save space during the seed
packaging process.
11 of 15
′ ; (c) PAW 20′ .
Figure 8.
Optical microscope
of Daucus
carota
at 500× magnification.
(a)(water);
Control(b)
(water);
Figure 8. Optical
microscope
images of images
Daucus carota
at 500
× magnification.
(a) Control
PAW 5(b)
PAW 5′; (c) PAW 20′.
Previous studies with the GAD reactor indicated that high concentrations of NOx can be
generated
theGAD
gas phase
andindicated
further transported
to the liquid phase,
where
compounds such
Previous studies
withinthe
reactor
that high concentrations
of NO
x can
as the
H2Ogas
also
appear
through
secondary
reactions
[50].
In
the
case
of
PAW
treatment
of seeds,
be generated in
phase
and
further
transported
to
the
liquid
phase,
where
2
these
can cause
an increase
in germination,
e.g.,[50].
through
H2O
accumulation
[48,49].
compounds such
as compounds
H2O2 also appear
through
secondary
reactions
In the
case
of
2
The
increase
in
germination
may
also
be
related
to
other
effects
of
plasma
treatment
on
seeds,
PAW treatment of seeds, these compounds can cause an increase in germination, e.g.,
such
as
surface
etching
and
increased
water
absorption
[67–69],
or
inactivation
of
harmful
through H2O2 accumulation [48,49]. The increase in germination may also be related to
these
also affect
seeds through
[16,70,71].
other effects of organisms
plasma treatment
on Many
seeds, of
such
as effects
surfacecan
etching
and increased
water active particles
activating [16,70,71].
the catalase
enzyme
for effects
the synthesis of new
found
PAW, e.g.,ofvia
H2 O2 organisms
absorption [67–69],
or in
inactivation
harmful
Many
of these
proteins
[26], increases
in chlorophyll
content
of hard seeds,
can also affect seeds
through
active particles
found in
PAW,[72],
e.g.,the
viacracking
H2O2 activating
the thus allowing
them
to
absorb
moisture
[73]
or
the
enhancement
of
nitrogen
contents
in
the seeds through
catalase enzyme for the synthesis of new proteins [26], increases in chlorophyll content
adsorption,
diffusion
and
trapping
of
RNS
produced
in
plasmas
[74].
Their
[72], the cracking of hard seeds, thus allowing them to absorb moisture [73] or the effect can not
be ancontents
increaseininthe
germination,
but adsorption,
also an increase
in theand
quality
of the plants grown
enhancement ofonly
nitrogen
seeds through
diffusion
trapping
and
their
levels
of
disease
tolerance
[75,76].
of RNS produced in plasmas [74]. Their effect can not only be an increase in germination,
Wethe
speculate
the
results
of seeds’
especially
with the presence
but also an increase in
quality that
of the
plants
grown
and imbibition
their levelsinofPAW,
disease
tolerance
of
RONS
and
low
pH,
changed
the
osmotic
potential
and
made
the
seed
surface more
[75,76].
prone to structural changes.
On the basis of the results, it can be concluded that when using the same reactor and
the same treatment conditions, the results largely depend on the type of treated seeds.
In the case of Beta vulgaris, the effect of PAW treatment is evident in both the change of
microflora, germination parameters and surface structure of the tested seeds. Due to the
higher rate of germination (GEN ), sprouts of greater length were obtained in addition to
a larger fraction of germinated seeds. Because of the relatively small decontamination
effect of fungi whose specific species were restricted or stimulated, the gradual increase
in treatment efficiency with increasing treatment time may be related to the change in
surface structure observed via optical and scanning electron microscopy. In the case of
Daucus carota, characterized by other types of fungi, a complete reduction in Alternaria
radicina and Trichoderma harzianum species, which were not removed during the classical
decontamination method with sodium hypochlorite, can be observed. Compared to the
control, significantly better germination and sprout length parameters were obtained
even for shortest time, but these were still worse than the results obtained for traditional
treatment with NaOCl. In combination with the overall better decontamination effect of
sodium hypochlorite, it can be thought that for carrot seeds, the decisive influence on
germination was connected with fungi on the surface, which is better removed by the
traditional method. However, the use of PAW may still offer a competitive alternative due
to the ecology and economy of plasma treatment with the investigated device, for which
only electricity and commonly available air are needed. PAW can be also generated in
larger quantities in devices of different constructions [77–79], which can further improve
the cost factor.
4. Conclusions
Plasma activated water positively influenced the seeds germination process; however,
differences between the plant species, in terms of the seeds’ response, were observed.
Longer PAW imbibition times resulted in better germination parameters in comparison
Appl. Sci. 2021, 11, 6164
12 of 15
to the control sample. For Beta vulgaris, treatment times of 10 and 20 min also allowed
a higher fraction of germinating seeds than NaOCl treatment, with only slightly shorter
average sprout lengths. For Daucus carota, the germination rate and sprout length were
both noticeably lower than for sodium hypochlorite solution, but still several times higher
compared to the control.
Plasma activated water had an impact on the composition of fungal species inhabiting
Beta vulgaris and Daucus carota seeds. Fungal species responded to the PAW treatment
in different manners; some of them were unaffected. The highest counts isolated were
assigned to the harmful producers of mycotoxins such as Alternaria alternata and moulds
of the genus Penicillium. The beneficial species were dominated by Epicoccum nigrum,
Clonostachys rosea and Trichoderma sp.
Author Contributions: Conceptualization: J.P., M.K. (Marek Kopacki) and K.H.; methodology: J.P.,
M.K. (Michał Kwiatkowski), P.T., M.K. (Marek Kopacki) and K.H.; investigation: J.P., P.T., M.K.
(Michał Kwiatkowski), M.K. (Marek Kopacki) and K.H. data curation: P.T., M.K. (Marek Kopacki)
and J.P., writing—original draft preparation: P.T. and J.P.; writing—review and editing: P.T., J.P. and
M.K. (Marek Kopacki); visualization: J.P., M.K. (Michał Kwiatkowski) and P.T.; supervision: J.P. All
authors have read and agreed to the published version of the manuscript.
Funding: This study was supported by the Polish–Slovak Bilateral Cooperation Programme (PlasmaBioAgro) PPN/BIL/2018/1/00065+SK-PL-18-0090 and the LUT research fund (FD-20/EE-2/418).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Acknowledgments: We would like to thank Eng. Łukasz Kaca for the technical assistance during
selected research tasks and Łukasz Remez from PIK INSTRUMENTS Sp.z.O.O for the SEM documentation. We acknowledge fruitful discussion with COST Action PlAgri CA19110 and CEEPUS
CIII-AT-0063 members.
Conflicts of Interest: The authors declare no conflict of interest.
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