Sterilizing Effect of Xanthomonas Campestris pv. Campestris (Xcc)
by Corona-Discharge Nonthermal Plasma Exposure at
Atmospheric Pressure
Li-li Dinga, Zi-mu Xub, Min-chen Wanga, Yong-yi Shena, Wei-dong Xiab
a
Department of Life Science, University of Science and Technology of China, Hefei, Anhui Province, China
b
Laboratory of Applied Plasma (APL), Department of Thermal Science and Energy Engineering, University of
Science and Technology of China, Hefei, Anhui Province, China
Abstract: Black rot disease of Chinese cabbage is caused by infection of Xanthomonas campestris
pv.Campestris (Xcc). It is also kind of worldwide familiar disease of crucifers. Lots of researches have been done
and the effect of nonthermal plasma exposure in sterilizing bacteria has been proved high-efficiency. In this
experiment, Xcc is purified from the cabbages infected with black rot disease, then disposed in AC
corona-discharge nonthermal plasmas at atmospheric pressure to investigate the sterilizing effect. From the result,
the sterilizing effect is obvious and it has a direct ratio with exposure time and corona current. Morphological
observation shows lysis of Xcc after the exposure and reasons are discussed following. Some cabbage seeds
soaked with Xcc are planted and exposed to make a further study of the idea of seed-sterilization by nonthermal
plasma.
Keywords: Sterilization, Xanthomonas campestris pv.Campestris (Xcc), nonthermal plasmas.
1. INTRODUCTION
B
LACK ROT, caused by the bacterium
Xanthomonas campestris pv. campestris (Xcc),
is considered the most important and most
destructive disease of crucifers, infecting all
cultivated varieties of brassicas worldwide [1],[2].
Host infection by Xcc can occur at any stage of the
plant life cycle. Characteristic symptoms of black rot
caused by Xcc are V-shaped chlorotic to necrotic
lesions extending from the leaf margins and
blackening of vascular tissues (Fig. 1.).
Figure 1. Xcc-infected brassicas (a. infected leaf of b.
campestris ssp. Pekinensis. b. infected leafstalk of leaf of b.
campestris ssp. Pekinensis. c. infected leaf of b. campestris ssp.
Chinensis. d. infected leaf of Chinese cabbage.)
There has been a recent resurgence in the study of
the gas discharge for disinfection technologies, and
more specifically using nonthermal plasma at
atmospheric pressure [4],[6]-[7]. Treatment with
such plasmas has been demonstrated to be a
powerful method of inactivating many different
types of microorganisms including Gram-negative
and Gram-positive bacteria, bacterial endospores,
yeasts, viruses and biofilms on the surfaces and in
aqueous solutions [3]-[5], [8]. The most common
plasma inactivation mechanisms that cause lethal
effects to microorganisms are UVC and VUV,
oxygen species, charged particles and heat.
1st circle: 6 needles, 1 needle per 60°, r= 1 cm;
2nd circle: 12 needles, 1 needle per 30°, r= 2 cm;
3rd circle: 24 needles,1 needle per 15°, r= 3 cm;
Radius of needlepoint: 0.1 mm;
Discharge frequency: 40 kHz.
3. Ground electrode: metal plate
Plasma treatment has many advantages in
comparison with other bacterial inactivation methods,
such as dry heat or hot steam sterilization, irradiation
by UV/gamma rays and other techniques [9]. Plasma
decontamination is usually fast, efficient, and safe in
terms of thermal, chemical or irradiation damage.
Therefore, in this paper, we try to investigate the
possibility of Xcc sterilization by nonthermal plasma
exposure in the direction of biophysics. For this
purpose, we purify Xcc from infected cabbage leaves
and establish a corona-discharge apparatus to
produce nonthermal plasma. Furthermore, cabbage
seeds soaked with Xcc are planted after plasma
exposure so as to examine the capability of plasma
sterilization to seeds as Xcc on seed is the main path
for Xcc infecting according to its life circle.
Figure 2. Structure of the plasma-producing system.
2. EXPERIMENTAL SETUP
A. Bacterial Sample Preparation
Figure 3. The vertical view of corona electrode.
1. Culture, separate and purify Xcc from infected
cabbage leaves.
3.EXPERIMENTAL METHOD
2. Identify: Penetrate the purified Xcc bacterial liquid
into healthy cabbage leaves to select the liquid which
causes typical symptoms of black rot disease.
A. Investigate Sterilizing Capacity of Xcc by
Nonthermal Plasma
Soak appropriate amount of healthy cabbage seeds in
Xcc bacterial liquid (10-6) for 12h.
Treat Xcc with nonthermal plasmas in current
(average) and time grads. Untreated control group is
established. Take micrographs to investigate the
morphological changes of treated Xcc and make
comparison with the untreated group.
C. Corona-discharge Nonthermal Plasma Producing
System
B. Investigate Sterilization of Cabbage Seeds with
Xcc by Nonthermal Plasma
1. Power supply properties
AC power supply; Vout= 0~15 kV; Fout= 15~45
kHz, changable;
2. Corona electrode (Fig. 3.)
The corona electrode is composed of 3
concentric circles of needles. 1 needle is taken as
the centre.
Cabbage seeds with Xcc are treated by nonthermal
plasma in current and time grads. Then cultivate
those seeds in sand and nutrient solution. Taking
notes of the quantity and quality of the germinating
seeds among all the groups respectively every day.
Cultivate cabbage seeds with Xcc as the control
group which are without nonthermal plasma
B. Infected Seeds Preparation
treatment.
4. RESULTS& DISCUSSION
A. Sterilizing Capacity of Xcc by nonthermal Plasma
In the experiment, dilute Xcc bacterial liquids of
different concentration (10-8,10-7,0.5*10-7) are spread
on respective medium plates and treated under
nonthermal plasmas produced on different current.
Sterilizing rate (%)
100
80
5 mA
7 mA
9 mA
-7
c= 0.5*10
60
40
20
0
0
1
2
3
4
5
Time (min)
Calculate the sterilization rate of Xcc. From Fig.4,
the sterilization rate when I=9 mA is found lower
than that of I=7 mA. Taking many relevant factors
and the properties of the plasma-producing apparatus
into consideration, breakdown between corona
electrode and the metal plate in limited space when
I=9 mA is supposed to be the reason. So, in later
parts of the experiment, the highest current has
adopted 7 mA.
The sterilization rate of Xcc has positive
correlation with current and exposure time as Fig. 4.
shows (I= 5 mA, 6 mA, 7 mA). The sterilization rate
of Xcc has reached approximately 90 % in the
condition of 7 mA and 5 min. So, It can be said that
the sterilizing efficiency of Xcc is relatively high by
means of direct nonthermal plasmas exposure.
B. Mophological Observation
Sterilizing rate (%)
100
80
5 mA
7 mA
9 mA
-8
c= 1*10
60
40
20
0
0
1
2
3
4
5
Time (min)
Comparing micrographs between plasma-treated
and untreated Xcc (Fig. 5a., Fig. 5b.), facts showed
that after plasma exposure, Xcc cells underwent
severe morphological changes such as lysis and
fusion while the untreated Xcc has a clear cell shape
with integrated edge. So, it can be firmly believed
that nonthermal plasmas have demolished the
bacterial structure of Xcc.
Sterilizing rate (%)
100
80
5 mA
6 mA
7 mA
-7
c= 0.5*10
60
40
20
0
0
1
2
3
4
5
Sterilizing rate (%)
Time (min)
100
90
80
70
60
50
40
30
20
10
0
-10
Figure 5. left. Micrographic image of natural Xcc.; right.
Micrographic image of plasma-treated Xcc.
C. The Sterilizing Effect of Xcc on Cabbage Seeds By
Nonthermal Plasma
1. There is none effect noticable on the germinating
5 mA
6 mA
7 mA
-7
c= 1*10
0
1
2
3
Time (min)
4
5
Figure 4. The sterilizing rate of Xcc by nonthermal plasma.
capacity of seed by nonthermal plasma exposure. It
is supposed to tell that plasma exposure is safe to the
seeds under compatible intensity.
2. The black rot symptoms are first spotted on young
seedlings on the 5th day of germination. During
germination, a part of seedlings become Xcc
infected.
3. The sterilizing effect of Xcc on cabbage seeds by
nonthermal plasma is approximately 50 % (7 mA, 5
min) and the survival rate of seeds increases along
with the enhance in plasma processing intensity. But
the sterilizing rate (as high as 50 %) is relatively low
in comparison with it which by plasma exposure on
medium in petri dish (as high as 90 %). So, it can be
assumed that in a well-designed device, the
sterilization of Xcc on seeds by nonthermal plasma
will be more efficient.
5. CONCLUSION
In this paper, the effect of Xcc neutralization on
medium and seeds by exposure of atmospheric
nonthermal plasma has been researched. According
to the experimentle results, the excellent sterilization
effect of Xcc by nonthermal plasma exposure has
been confirmed. So, it is a brand new method for
prevention and cure of the black rot disease, which is
caused by Xcc--the most important and most
destructive disease of crucifers, infecting all
cultivated varieties of brassicas worldwide.
What is the main reaction product and the
dominant species in this corona-discharge
atmospheric nonthermal plasma is still unknown.
Only further research will elucidate the extent to
which the mechanisms discussed in the introduction
are involved in the bactericidal action and dominate.
Biological and chemical challenges are still to be
overcome in the development of effective
atmospheric pressure discharges for disinfection of
Xcc. On the biological side, it remains critical to
understand the pathway of damage leading to death
for Xcc. Without this knowledge, optimization of the
disinfection process is reduced to an empirical affair.
In elucidating the processes leading to death of Xcc,
the actual cause must be separated from any physical
changes that occur postmortem. One approach would
be to study the sublethal effects of plasma on Xcc,
while the effects on individual cellular components
such as cell membranes, nucleic acids, poteins, and
enzymes could also be considered. On the chemical
side, a deeper appreciation regarding the relative
importance of the various reactive species involved
in bacterial neutralization is urgently needed. It is
clear that a truly multidisciplinary approach is
needed in order to fully understand the biophysical
and biochemical processes [10]. The sterilizing
efficiency of Xcc by nonthermal plasma exposure at
atmospheric pressure will be highly enhanced after
figuring them out.
References
[1] Alvarez AM. "Black rot of crucifers." In: Slusarenko
AJ, Fraser RSS, van Loon LC (Eds.) Mechanisms of
Resistance to Plant Diseases. Dordrecht, The
Netherlands: Kluwer Academic Publishers, 2000. pp
21-52.
[2] Williams PH. "Black rot: a continuing threat to
world crucifers." Plant Disease 64.8 (1980):
736-742.
[3] S. Lerouge, M. R. Wertheimer, and L. H. Yahia,
“Plasma sterilization: A review of parameters,
mechanisms, and limitations,” Plasmas Polym., vol. 6,
pp. 175–188, 2001.
[4] M. Laroussi, “Non-thermal decontamination of
biological media by atmospheric pressure plasmas:
Review, analysis and prospects,” IEEE Trans.
Plasma Sci., vol. 30, pp. 1409–1415, Aug. 2002.
[5] H.W.Herrmann, I.Henins, J. Park, and G. S. Selwyn,
“Decontamination of chemical and biologicalwarfare
(CBW) agents using and atmospheric pressure
plasma jet,” Phys. Plasmas, vol. 6, no. 5, pp.
2284–2289, 1999.
[6] B. R. Gadri, J. R. Roth, T. C. Montie, K.
Kelly-Wintenberg, P. P. Y. Tsai, D. J. Helfritch, P.
Feldman, D.M. Sherman, F. Karakaya, and Z. Y.
Chen, “ Sterilization and plasma processing of
room temperature surfaces with a one atmosphere
uniform glow discharge plasma (OAUGDP),” Surf.
Coat. Technol., vol. 131, no. 1–3, pp. 528–542, Sep.
2000.
[7] T. C. Montie, K. Kelly-Wintenberg, and J. R. Roth,
“An overview of research using the one atmosphere
uniform glow discharge plasma (OAUGDP) for
sterilisation of surfaces and materials,” IEEE Trans.
Plasma Sci., vol. 28, no. 1, pp. 41–50, Feb. 2000.
[8]
M. Laroussi, I. Alexeff, and W. L. Wang,
“Biological decontamination by nonthermal
plasma,” IEEE Trans. Plasma Sci., vol. 28, no. 1,
pp. 184–188, Feb. 2000.
[9] S. Lerouge, M. R. Wertheimer, and L. H. Yahia,
“Plasma sterilization: A review of parameters,
mechanisms, and limitations,” Plasmas Polym., vol.
6, pp. 175–188, 2001.
[10] Lindsey F. Gaunt, Clive B. Beggs, and George E.
Georghiou, “Bactericidal action of the reactive
species produced by gas-discharge nonthermal
plasma at atmospheric pressure: a review,” IEEE
Trans. Plasma Sci., vol. 34, no. 4, pp. 1257-1269,
Aug. 2006.