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Biophysical control of the growth of Agrobacterium tumefaciens using extremely low frequency electromagnetic waves at resonance frequency
Biochemical and Biophysical Research Communications 494 (2017) 365e371
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Biophysical control of the growth of Agrobacterium tumefaciens using extremely low frequency electromagnetic waves at resonance frequency M. Ali Fadel a, Reem H. El-Gebaly a, Shaimaa A. Mohamed a, Ashraf M.M. Abdelbacki b, * a b
Biophysics Department, Faculty of Science, Cairo University, Egypt Plant Pathology Department, Faculty of Agriculture, Cairo University, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 September 2017 Accepted 2 October 2017 Available online 4 October 2017
Isolated Agrobacterium tumefaciens was exposed to different extremely low frequencies of square amplitude modulated waves (QAMW) from two generators to determine the resonance frequency that causes growth inhibition. The carrier was 10 MHz sine wave with amplitude ±10 Vpp which was modulated by a second wave generator with a modulation depth of ± 2Vpp and constant field strength of 200 V/m at 28 C. The exposure of A. tumefaciens to 1.0 Hz QAMW for 90 min inhibited the bacterial growth by 49.2%. In addition, the tested antibiotics became more effective against A. tumefaciens after the exposure. Furthermore, results of DNA, dielectric relaxation and TEM showed highly significant molecular and morphological changes due to the exposure to 1.0 Hz QAMW for 90 min. An in-vivo study has been carried out on healthy tomato plants to test the pathogenicity of A. tumefaciens before and after the exposure to QAMW at the inhibiting frequency. Symptoms of crown gall and all pathological symptoms were more aggressive in tomato plants treated with non-exposed bacteria, comparing with those treated with exposed bacteria. We concluded that, the exposure of A. tumefaciens to 1.0 Hz QAMW for 90 min modified its cellular activity and DNA structure, which inhibited the growth and affected the microbe pathogenicity. © 2017 Elsevier Inc. All rights reserved.
Keywords: Agrobacterium tumefaciens Growth inhibition Electromagnetic waves Resonance frequency
1. Introduction Agrobacterium tumefaciens causes crown gall disease on a wide range of dicotyledonous host species especially members of the rose family such as apple, pear, peach, cherry, almond, raspberry and roses. Basically, part of the bacterium DNA (the T-DNA) is transferred to the plant, which integrates into genome of the plant, causing tumors production and associated changes in plant metabolism [1] resulting in economic loss of plant yield. For treatment of plant infection, many trials have been carried out all over the world to control the disease without much success. No promising control was achieved using antibiotics, soil fumigants, chemical control [2], or breeding of resistant varieties [3,4]. Therefore, several studies have been performed to show the possible effects of electric, magnetic and electromagnetic fields on bacterial growth as alternative methods for the treatment of bacterial infections [5,6].
* Corresponding author. E-mail address: [email protected] (A.M.M. Abdelbacki). https://doi.org/10.1016/j.bbrc.2017.10.008 0006-291X/© 2017 Elsevier Inc. All rights reserved.
Recently, extremely low electromagnetic waves of very low field intensity which resonates with bioelectric signals generated during a particular metabolic activity are used to control the cellular activity of microorganisms. These experiments succeeded to control the growth of Ehrlich tumors in mice [7,8], fungi [9] and bacteria [10,11]. Therefore, the aim of the present study was to find out the resonance frequency of the electromagnetic waves that inhibit the activity of A. tumefaciens and overlap its ability to make division, as well as to determine the changes that may occur at the molecular level as a result of exposure to electromagnetic fields (ELF-EMFs). 2. Materials and methods 2.1. Microorganism growth conditions The strain of A. tumefaciens (ATCC 19358) used in this study was obtained from Microbiological Resources Center (Cairo MIRCEN), Faculty of Agriculture, Ain Shams University, Egypt. Subculture broth media was prepared by inoculating a test tube containing 5 ml sterile nutrient broth/pH 7.1 (Biolife, Milan, Italy) with two
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single colonies of bacteria, then incubated at 28 C for 48 h.
was considered.
2.2. Bacterial reproduction
2.8. Dielectric measurements
The Bacterial growth of A. tumefaciens was determined by measuring the absorbance at 600 nm (A600) every 1 h using spectrophotometer model (6405 UV/Vis) manufactured by Jenway in UK.The absorbance of bacterial suspension then plotted as a function of incubation time.
Bacterial suspensions were subjected to centrifugation at 14,000 rpm at 4 C for 15 min, the pellets were taken and resuspended in a 1 ml volume of sterile deionized water, then the tubes were centrifuged and the pellets were washed with deionized water twice more, finally the pellets were re-suspended in sterile deionized water. A fixed concentration of bacterial cells (107 cfu/ml) was used. The dielectric measurements were done for all samples in the range frequency (f) 0.1e4 MHz using a Loss Factor Meter (type HIOKI 3532 LCR Hi TESTER, version 1.02, Japan), using a sample cell (PW 9510/60; Philips, Weisshausstrasse, Aachen, Germany). The sample cell has two squared platinum black electrodes of area (A) 0.64 cm2 each with an inter-electrode distance (d) of 1 cm. During measurements, the sample between the electrodes was kept at a constant temperature of 24 ± 0.1 C. The capacitance (C) of the samples was measured at each frequency and the resistance was recorded. Each run was taken three times and the average was calculated. The relative permittivity ὲ, loss tangent 00 “tan d”, dielectric loss ε , conductivity s and relaxation time (t) of the samples were calculated for each frequency using the following relation:
2.3. Count-absorbance calibration curve A standard calibration curve was constructed between the absorbance of different concentrations of bacterial suspension and the number of colony forming units (CFU) per ml which determined by plate counting technique [12]. 2.4. ELF EM exposure system Samples of A. tumefaciens suspension were exposed to different extremely low frequencies of square amplitude modulating waves (QAMW). The modulating waveform was square and the carrier frequency was 10 MHz sine wave. The wave carrier was generated by a wave generator model AFG 310 manufactured by Sony Tektronics, Japan, and the modulating wave was generated by synthesized arbitrary generator type TTi TGA1230 manufactured by Thurlby Thandar Instruments Ltd, England. The amplitude of the wave carrier was 10 Vpp and the modulating depth was ±2 V. Different groups of A. tumefaciens were exposed to different frequencies of the QAMW through two parallel cupper electrodes of dimensions 5 5 cm2 and separation distance 1 cm with constant field strength of 200 V/m.
ε0 ¼ Cd=ε0 A tand ¼
(1)
. 00 1 pfRC ¼ ε ε0 2
(2)
00
s ¼ 2pf,ε ε0
(3)
1 pf c 2
(4)
2.5. Determination of resonance frequency of growth inhibition
T¼
The A. tumefaciens suspension was divided into thirteen groups (each group contains three samples of A. tumefaciens suspension), one group kept as control and the other twelve groups exposed to different frequencies of QAMW in the range from 0.1 to 1.3 Hz in steps of 0.1 Hz for a period of 60 min. At the end of the exposure period, the control and exposed groups were incubated at 28 C and every 1 h the incubations were interrupted for absorbance measurements at 600 nm in order to determine the resonance frequency of growth inhibition.
Where, εo is the permittivity of free space, fc is the frequency at the midpoint of the dielectric dispersion curve and R is the resistance of the specimen in Ohm.
2.6. Determination of the optimum exposure time Eight groups of A. tumefaciens suspension were prepared; one kept as control and the other groups were exposed to QAMW at the inhibiting resonance frequency for periods (60, 90, 120 and up to 150 min). At the end of the exposure time; the samples were incubated at 28 C. Every 1-h the samples were shaken and the absorbance were measured then the corresponding number of cells was calculated from the calibration curve. 2.7. Antibiotic sensitivity test A. tumefaciens was subjected to susceptibility test using the following antimicrobial agents: Amikacin 30 mg (AK), Gatifloxacin 5 mg (GAT), Ciprofloxacin 5 mg (CIP), Carbenicillin 100 mg (PY), Chloramphenicol 30 mg (C), Gentamicin 10 mg (CN), Cefaclor 30 mg (CEC), and Rifampin 5 mg (RA). Antimicrobial sensitivity test was carried out and performed by the procedure outlined by [13]. The sensitivity test was carried three times for each of the treated samples and the control, then the average value for the readings
2.9. DNA analysis The DNA extraction was made from 50 mg of fresh culture of A. tumefaciens according to the method modified and developed by [14]. The quality and quantity of the DNA was measured by means of both agarose gel electrophoresis and spectrophotometer. Code and nucleotide sequence of tested primers used in the random amplified polymorphic DNA (RAPD) reactions is shown in Table 1. RAPD reactions were performed according to [15]. RAPD reactions were conducted in a Techne TC-412 thermocycler (Barloworld Scientific Ltd, United Kingdom). Mid-range DNA Ladder (100e3000 bp) (Jena Bioscience, place, Germany) was used and gel documentation system (AAB Advanced American Biotechnology 1166 E. Valencia Dr. Unit 6 C, Fullerton, CA 92631) and The similarity level was determined by un-weighted pair group method based on arithmetic mean (UPGMA).
Table 1 RAPD primer: names and sequences. Primer name
Primer sequence
OPA-02 OPA-10 OPA-13 OPK-08 OPK-10
50 -TGCCGAGCTG-30 50 -GTGATCGCAG-30 50 -CAGCACCCAC-30 50 -GAACACTGGG-30 50 -GTGCAACGTG-30
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2.10. Transmission electron microscope (TEM) examination The morphological changes of A. tumefaciens due to the exposing to QAMW at resonance frequency for the most effective time have been determined using TEM (JEM-1400; JEOL Ltd., Akishima, Tokyo, Japan) presented at Faculty of Agriculture Research Park-Cairo University. The bacterial samples passed through some processing according to [16]. Images were captured by CCD camera model AMT, optronics camera with 1632 1632 pixel format as side mount configuration. 2.11. The pathogenicity test of A. tumefaciens strain To determine the pathogenicity of A. tumefaciens strains before and after exposing to QAMW; 10 ml of bacterial suspension has a concentration of 109 CFU/ml was prepared and divided equally into two groups, one group kept as control and the other one exposed to QAMW at resonance frequency of growth inhibition for the most effective time. Healthy tomato plants were planted and divided into three groups, each group contained five plants. A pin prick was then made into the stems of all plants and three drops of sterile deionized water were placed at the wound sites in the stem of the first group. The second group was infected with non-exposed bacterial suspension and the last group was infected with exposed bacterial suspension. Symptoms were then followed-up after inoculation and the photographs were taken 4e6 weeks later. 2.12. Statistical analysis The statistical analysis was performed using student's t-test and ANOVA analyses, with a minimal confidence level of 0.05 for statistical significance. Each experimental study was performed at least three times with a minimum of three samples per termination point.
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1 h (P < 0.01). From the standard calibration curve, the number of CFU/ml at stationary phase for the control and group exposed to 1.0 Hz QAMW for 1hr was calculated as shown in Table 2. It is clear from the data that the population intensity of the exposed samples had been reduced as compared to that of the control with highly significant inhibition (P < 0.01).
3.2. Effects of exposure time The growth inhibition percentages of A. tumefaciens after the exposure to 1.0 Hz QAMW for different exposure periods at 12 h of incubation are shown in Table 3. The results showed that 90 min exposure gave the maximum growth inhibition at the resonance frequency (49.2%).
3.3. antibiotic susceptibility test The antibiotic susceptibility test results for control and exposed to 1.0 Hz QAMW samples are given in Table 4. It is clear that exposure to QAMW at 1.0 Hz for 90 min (the most effective time) gave significant increase in the sensitivity of A. tumefaciens to all antimicrobial agents used.
3.4. Effects on the molecular structure 3.4.1. Dielectric relaxation Table 5, represented the values of the relaxation time (t), the dielectric increment (Dέ) and the electric conductivity (s) for control and exposed group. The data obtained indicated pronounced decrease in the relaxation time, dielectric increment and conductivity for the exposed sample compared with the control ones.
3. Results 3.1. Growth curve characteristics of A. tumefaciens Fig. 1, showed the differences in absorbance between bacterial groups exposed to QAMW in the range 0.0 Hz up to 1.3 Hz for 1 h and control group at 12 h incubation time as a function of the applied frequency. The data indicated a highly significant growth inhibition occurred after exposure to 1.0 Hz QAMW for
3.4.2. DNA analysis Three out of five screened primers provided informative RAPD pattern for all isolates. Fig. 2a, indicates the appearance of new band in the exposed samples at 490 bp for primer OPA-10, however, Fig. 2b and c, showed disappearance of 3 bands in the exposed samples; at 750, 420 bp, for OPA-13 primer and at 900 bp for OPK08 primer. There is no difference in RAPD patterns for both control and exposed samples appeared for primers OPA-02 and OPK-10.
Fig. 1. The change in absorbance post 12 h of incubation with respect to control as a function of frequency.
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M.A. Fadel et al. / Biochemical and Biophysical Research Communications 494 (2017) 365e371 Table 2 The concentration of A. tumefaciens in (cfu/ml) before and after exposure to 1.0 Hz QAMW for 1hr with its inhibition percentage difference. Sample
N at the stationary phase x 108 (cfu/ml)
Growth Inhibition Percentage G%
Control 1.0 Hz QAMW
(14.6 ± 0.3) (11.2 ± 0.21)
0% (23.3%)**
NS
Not Significant. Significant (P < 0.05). Highly Significant (P < 0.01). *** Very High Significant (P < 0.001). *
**
Table 3 The growth inhibition percentages of A. tumefaciens exposed to 1.0 Hz QAMW for different exposure periods at 12 h of incubation. Exposure time (min) Growth inhibition percentage (G %)
30 (10.7%)*
45 (14.5%)*
60 (23.3%)**
75 (32.2%)**
90 (49.2%)**
120 (30.4%)**
150 (29%)**
NS
Not Significant. Significant (P < 0.05). Highly Significant (P < 0.01). *** Very High Significant (P < 0.001). *
**
Table 4 The mean of the inhibition zones diameter of different antimicrobial agents for control and sample exposed to 1.0 Hz QAMW for 90 min. Frequency
Antibiotic Mean inhibition zone diameter (mm) C
CN
AK
Inhibitors for protein synthesis Control 1.0 Hz QAMW
(26.5 ± 0.2) (35 ± 0.3)**
(23 ± 0.26) (30 ± 0.2)**
(23.5 ± 0.4) (33 ± 0.32)***
GAT
CIP
PY
CEC
RA
Inhibitors for cell wall synthesis
Inhibitors for DNA
Inhibitors for RNA synthesis
(25 ± 0.3) (34 ± 0.2)***
No effect (11 ± 0.13)**
(12 ± 0.36) (25 ± 0.41)***
(24 ± 0.12) (32 ± 0.24)**
No effect (13 ± 0.42)**
NS
Not Significant. Significant (P < 0.05). Highly Significant (P < 0.01). *** Very High Significant (P < 0.001). *
**
Table 5 The relaxation time (t), dielectric increment (Dέ) and conductivity (s) of control and exposed to 1.0 Hz QAMW for 90 min samples. Sample/Dielectric parameter
Relaxation time (t) (107sec)
Dὲ¼(ὲ0 - ὲ∞)
Conductivity (s) at 4 MHz (x107 S/m)
Control (unexposed) 1.0 Hz QAMW
(0.49 ± 0.24) (0.31 ± 0.72)*
(246 ± 9.21) (166 ± 2.75)***
(56 ± 3.57) (43 ± 1.53)**
NS
Not Significant. Significant (P < 0.05). ** Highly Significant (P < 0.01). *** Very High Significant (P < 0.001). *
Fig. 2. Electrophoretic RAPD patterns (a) OPA-10 primer, (b) OPA-13 primer and (c) OPK-08 primer. M ¼ DNA Ladder (DNA Marker). C ¼ DNA of the control sample. E ¼ DNA of the treated sample exposed to 1.0 Hz QAMW for 90 min.
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3.5. Bacterial images by TEM
4. Discussion
Fig. 3a and b showed TEM images for the control A. tumefaciens cells. The majority of the cells have a well preserved cell envelope that consists of a smooth, evenly stained plasma membrane, a semi translucent periplasmic gel and a symmetrically stained, taut outer membrane. Additionally, the cells look remarkably robust and their chromosome is spread throughout the entire cytoplasm over the binary fission which confirming an active metabolic processes. Images of exposed cells to 1.0 Hz QAMW are shown in Fig. 3c and d; where, fragmentation of DNA, disruption and disintegration of cell wall and cytoplasmic membrane, an extrusion of cytoplasmic contents from cell wall and abnormal septation were observed.
The present work introduced a new method for controlling the crown gall disease via inhibition of A. tumefaciens growth using extremely low frequency electromagnetic fields (ELF-EMF) at resonance frequency of growth inhibition. The present findings indicated that the exposure to 1.0 Hz QAMW for 1 h caused highly significant growth inhibition for A. tumefaciens (Fig. 1 and Table 2) and confirming that it is the resonance frequency of growth inhibition for this bacteria. The inhibition effect of the ELF-EMF is due to the interference of this field according to its frequency with the bioelectric signals generated from physiological functions of bacterial cells. The results of these interference reactions depend on the mode of interference pattern which may lead to inhibition (destructive mode) or enhancement (constructive mode) to the running physiological process [17]. The exposure of bacteria to ELFEMF is associated with many biological effects include; changes on cellular division and cell morphotype [18], DNA and gene expression alterations [19,20] protein synthesis [21] and transport of ions by cell membranes [22]. To illuminate the induced changes that may be occurred in the cellular membrane structure as a result of exposure to QAMW at resonance frequency two experiments were done, a) The dielectric properties and b) Antibiotic sensitivity test. The dielectric relaxation measurements as shown in Table 5, indicated pronounced decrease in the average values of the dielectric increment (Dέ), the relaxation time (t) and the electrical conductivity (s) for exposed bacterial samples. Of note, the electrical conductivity and relaxation time are directly related to the electric dipole moment of the macromolecule, which in turn dependent on
3.6. In vivo results As shown in Fig. 4aee, the symptoms of crown gall disease appeared on the crown and stems of infected plants 4e6 weeks after inoculation by unexposed bacteria. The symptoms include; sudden wilting of the plant, large tumors-like swellings (galls), droopy appearance of branches and the branches gradually turned bronze and die (Fig. 4b). It was found that the tumors were larger in size and more in number, and all pathological symptoms were very aggressive in tomato plants treated with non-exposed bacteria (Fig. 4d) comparing with those treated with exposed bacteria (Fig. 4c and e), which seemed to be quite similar to the healthy plants in Fig. 4a.
Fig. 3. TEM images of (a) Normal A. tumefaciens. (b) Normal A. tumefaciens cells during the binary fission. (c) Exposed A. tumefaciens cells showing abnormal septation (arrow 1), pores were formed inside the cell and in cell membrane and disorganization of cell contents during the cell binary fission (arrows 2, 3 and 4). (d) Exposed A. tumefaciens showed the dissolution of the cell wall and an extrusion of cytoplasmic contents from cell wall.
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Fig. 4. Symptoms on the plants 4e6 weeks after inoculation: (a) Healthy plants treated with deionized water, (b) plants treated with non-exposed bacteria, (c) plants treated with exposed bacteria, (d) plants treated with non-exposed bacteria and (e) plants treated with exposed bacteria to 1.0 Hz QAMW for 90 min.
its size and charge. This decrease in the dielectric parameters after exposure to ELF-EMF may be due to that the exposure of bacterial cells to these fields is associated with heat shock response (HSR) [23,24]. This HSR causes decrease in dielectric permittivity and cell polarizability [25]; and hence affect the cellular charges distribution. In view of the potential role of ELF-EMF in modulating charge movements on the membrane, it has been verified that ELF-EMF can affect membrane functions not only by local effects on ligand binding or ion fluxes but also by altering the distribution and/or the aggregation of the intra membrane proteins [26]. This analysis is supported by the data obtained from antibiotic sensitivity test results using different antimicrobial agents of different interaction mechanisms with the bacteria. As shown in Table 4, the highly significant increase in the sensitivity of A. tumefaciens after the exposure to the antibiotics AK, C and CN that are inhibitors for protein synthesis may be due to an enhancement in the penetration of aminolglycosides into bacteria which are cationic antibiotics that bind to anionic components of the bacterial cell membrane in a reversible and concentration-dependent manner [27]. In addition, the susceptibility increase of the microorganism to cell wall synthesis inhibitors (PY and CEC) after exposure may be due to the effect of electromagnetic waves on the enzymatic activity involved in the bacterial cell wall synthesis. This analysis are supported by TEM images, where, the microbial cells exposed to 1.0 Hz QAMW showed disruption and disintegration of cell wall and cytoplasmic membrane which permitted an extrusion of cytoplasmic contents. All these results support each other and can lead to the conclusion that exposure of A. tumefaciens to 1.0 Hz QAMW caused changes in the properties of cell structure which may affect the cellular activity of the microorganism and cell to cell communication. The appearance of new band in the amplified DNA in the exposed sample prove that the DNA sequences have been changed under the effect of QAMW in a way that the primer used find a new binding sequence which not present in the control sample. Also the absence of bands indicated that the genetic sequences of bacterial DNA were modified due to the exposure to 1.0 Hz QAMW. These DNA
mutations and genetic alterations resulted in changes in the translated proteins and enzymes involved in bacterial growth process and divisions [28,29]. These explain the highly significant increase in the sensitivity of A. tumefaciens after the exposure to the antibiotics CIP and GAT which are inhibitors for DNA after the exposure to 1.0 Hz QAMW. The in-vivo results showed that the symptoms of crown gall were more aggressive in plants treated with non-exposed bacteria comparing with those treated with exposed bacteria, which seemed to be quite similar to the healthy plants. The crown gall disease was appeared with very slight symptoms in the plants treated by exposed bacteria and almost all plants treated by non-exposed bacteria were hit by aggressive symptoms of crown gall disease. Based on these finding, it could be suggested that the exposure to ELF-EMF at the inhibiting resonance frequency to cellular growth affect the physiological behavior and pathogenic genes of A. tumefaciens, which in turn affect the pathogenicity of the bacteria as confirmed by DNA and in-vivo results. All of these changes can cause the weakening of the exposed bacterial cells, which in turn gave a chance to the defense mechanism of plant to overcome the pathogen and prevent the disease. We concluded from the present findings that the exposure of A. tumefaciens to QAMW at the inhibiting resonance frequency is a new promising technique as an aid to avoid the use of traditional disease control techniques or the use of antibiotics and soil fumigants to control the crown gall disease. Declaration of interest This work is a part of the Ph.D. thesis of Shaimaa Abd El-Raof Mohamed submitted to the Biophysics Department, Faculty of Science, Cairo University. No external funding was given and/or available to run this work. Conflicts of interest None.
M.A. Fadel et al. / Biochemical and Biophysical Research Communications 494 (2017) 365e371
References [1] M.D. Chilton, M.H. Drummond, D.J. Merio, et al., Stable incorporation of plasmid DNA into higher plant cells: the molecular basis of crown gall tumorigenesis, Cell 11 (1977) 263e271. [2] S. Murakoshi, M. Takahashi, Trials of some control of tomato wilt caused by Pseudomonas solanacearum, Bull. Kanagawa Hortic. Exp. Stn. 31 (1984) 50e56. [3] G. Hartman, J. Elphinstone, Advances in the control of Pseudomonas solanacearum race 1 in major food crops, in: A. Hayward, G. Hartman (Eds.), Bacterial Wilt: the Disease and its Causative Agent, Pseudomonas Solanacearum, CAB International, Wallingford, UK, 1994, pp. 157e178. [4] M. Lopez, E. Biosca, Potato bacterial wilt management: new prospects for an old problem, in: C. Allen, P. Prior, A.C. Hayward (Eds.), Bacterial Wilt Disease and the Ralstonia Species Complex, APS Press, St. Paul, Minnesota, USA, 2004, pp. 205e224. [5] Z. Liang, Z. Cheng, G. Mittal, Inactivation of spoilage microorganisms in apple cider using a continuous flow pulsed electric field system, LWT 39 (2006) 350e356. [6] I. Ayse, A. Burak, A. Zafer, A. Dilek, A. Nilufer, S. Tangul, Effect of extremely low frequency electromagnetic fields on growth rate and morphology of bacteria, Int. J. Radiat. Biol. (2011) 1e8. [7] M.A. Fadel, R. El-Gebaly, A. Aly, A. Sallam, O. Sarhan, H. Eltohamy, Preventing of Ehrlich tumor metastasis in liver, kidney and spleen by electromagnetic field, Int. J. Phys. Sci. 5 (2010) 2057e2065. [8] M.A. Fadel, R. El-Gebaly, A. Hamad, Combination of bacteriolytic therapy with magnetic field for Ehrlich tumour treatment, Gen. Physiol. Biophys. 36 (2017) 259e271. [9] M.A. Fadel, M.A. Ahmed, M.A. El Hag, Control of Sclerotium cepivorum (Allium White Rot) activities by electromagnetic waves at resonance frequency, Aust. J. Basic & Appl. Sci. 3 (2009) 1994e2000. [10] M.A. Fadel, S. Mohamed, A.M. Abdelbacki, A. El-Sharkawy, Inhibition of Salmonella typhi growth using extremely low frequency electromagnetic (ELFEM) waves at resonance frequency, J. Appl. Microbiol. 117 (2014) 358e365. [11] M.A. Fadel, R. Elgebaly, M. Elneklawi, A. Othman, Role of duty cycle on Pseudomonas aeruginosa growth inhibition mechanisms by positive electric pulses, Bio-Med. Mat. Eng 27 (2016) 211e225. [12] R. Atlas, Principles of Microbiology, Mosby- Year book, INC, 1995. [13] National Committee for Clinical Laboratory Standards (Ed.), Performance Standards for Antimicrobial Disk Susceptibility Tests, third ed., National Committee for Clinical Laboratory Standards Villanova, Pa, 1985. [14] S.L. Dellaporta, J. Wood, J.B. Hicks, A plant DNA minipreparation: version II, Plant Mol. Biol. Report. 1 (1983) 19e21.
371
[15] J.G.K. Williams, A.R. Kubelik, K.J. Livak, J.A. Rafalski, S.V. Tingey, DNA polymorphisms amplified by arbitrary primers are useful as genetic markers, Nucleic Acids Res. 18 (1990) 6531e6535. [16] M. Demicheli, A. Goes, A. Ribeiro, Ultrastructural changes in Paracoccidioides brasiliensis yeast cells attenuated by gamma irradiation, J. Compil. 50 (2007) 397e402. [17] M.A. Fadel, H.E. Reem, A.A. Amany, F.I. Fakhry, Control of Ehrlich tumor growth in mice by electromagnetic wave at resonance frequency, in vivo, Electromagn. Biol Med 24 (2005) 9e12. [18] L. Cellini, R. Grande, E. Di Campli, et al., Bacterial response to the exposure of 50 Hz electromagnetic fields, Bioelectromagnetics 29 (2008) 302e311. [19] J. Galvanoskis, J. Sandblom, Periodic forcing of intracellular calcium oscillators Theoretical studies of the effects of low frequency fields on the magnitude of oscillations, Bioelectrochem Bioenerg. 2 (46) (1999) 161e174. [20] A. El May, S. Snoussi, N. Ben Miloud, et al., Effects of static magnetic field on cell growth, viability, and differential gene expression in Salmonella, Foodborne Pathog. Dis. 6 (2009) 547e552. [21] E.M. Goodman, B. Greenebaum, M.T. Marron, Altered protein synthesis in a cell-free system exposed to a sinusoidal magnetic field, Biochim. Biophys. Acta 1202 (1993) 107e112. [22] G. D'Inzeo, S. Pisa, L. Tarricone, Ionic channel gating under electromagnetic exposure: a stochastic model, Bioelectrochem Bioenerg. 29 (3) (1993) 289e304. [23] O. Smith, Cells, stress and EMFs, Nat. Med. 2 (1996) 23e24. [24] B. Del Re, P. Marcantonio, F. Bersani, G. Giorgi, Extremely low frequency magnetic field exposure affects DnaK and GroEL expression in E. coli cells with impaired heat shock response, Gen. Physiol. Biophys. 28 (2009) 420e424. [25] Z. Boris, Z. Zhihui, N. Aphakorn, J. Ronald, Rapid microbiological testing: monitoring the development of bacterial stress, PLoS One 5 (10) (2010) 1e8 e13374. [26] A. Marchionni, M. Paffi, P. Pellegrino, Comparison between low-level 50 Hz and 900MHz electromagnetic stimulation on single channel ionic currents and on firing frequency in dorsal root ganglion isolated neurons, Biochimica Biophysica Acta 1758 (5) (2006) 597e605. [27] H.W. Taber, J.P. Mueller, P.F. Miller, A.S. Arrow, Bacterial uptake of aminoglycoside antibiotics, Microbiol. Rev. 51 (1987) 439e457. [28] J. Van der Wolf, J. Van Beckhoven, E. De Haan, G. Van den Bovenkamp, G. Leone, Specific detection of Ralstonia solanacearum 16S rRNA sequences by AmpliDet RNA, Eur. J. Plant Pathology 110 (2004) 25e33. [29] Y. Lee, S. Fan, L. Chiu, K. Hsia, Isolation of an insertion sequence from Ralstonia solanacearum race 1 and its potential use for strain characterization and detection, Appl. Environ. Microbiol. 67 (2001) 3943e3950.
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Biophysical control of the growth of Agrobacterium tumefaciens using extremely low frequency electromagnetic waves at resonance frequency M. Ali Fadel a, Reem H. El-Gebaly a, Shaimaa A. Mohamed a, Ashraf M.M. Abdelbacki b, * a b
Biophysics Department, Faculty of Science, Cairo University, Egypt Plant Pathology Department, Faculty of Agriculture, Cairo University, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 September 2017 Accepted 2 October 2017 Available online 4 October 2017
Isolated Agrobacterium tumefaciens was exposed to different extremely low frequencies of square amplitude modulated waves (QAMW) from two generators to determine the resonance frequency that causes growth inhibition. The carrier was 10 MHz sine wave with amplitude ±10 Vpp which was modulated by a second wave generator with a modulation depth of ± 2Vpp and constant field strength of 200 V/m at 28 C. The exposure of A. tumefaciens to 1.0 Hz QAMW for 90 min inhibited the bacterial growth by 49.2%. In addition, the tested antibiotics became more effective against A. tumefaciens after the exposure. Furthermore, results of DNA, dielectric relaxation and TEM showed highly significant molecular and morphological changes due to the exposure to 1.0 Hz QAMW for 90 min. An in-vivo study has been carried out on healthy tomato plants to test the pathogenicity of A. tumefaciens before and after the exposure to QAMW at the inhibiting frequency. Symptoms of crown gall and all pathological symptoms were more aggressive in tomato plants treated with non-exposed bacteria, comparing with those treated with exposed bacteria. We concluded that, the exposure of A. tumefaciens to 1.0 Hz QAMW for 90 min modified its cellular activity and DNA structure, which inhibited the growth and affected the microbe pathogenicity. © 2017 Elsevier Inc. All rights reserved.
Keywords: Agrobacterium tumefaciens Growth inhibition Electromagnetic waves Resonance frequency
1. Introduction Agrobacterium tumefaciens causes crown gall disease on a wide range of dicotyledonous host species especially members of the rose family such as apple, pear, peach, cherry, almond, raspberry and roses. Basically, part of the bacterium DNA (the T-DNA) is transferred to the plant, which integrates into genome of the plant, causing tumors production and associated changes in plant metabolism [1] resulting in economic loss of plant yield. For treatment of plant infection, many trials have been carried out all over the world to control the disease without much success. No promising control was achieved using antibiotics, soil fumigants, chemical control [2], or breeding of resistant varieties [3,4]. Therefore, several studies have been performed to show the possible effects of electric, magnetic and electromagnetic fields on bacterial growth as alternative methods for the treatment of bacterial infections [5,6].
* Corresponding author. E-mail address: [email protected] (A.M.M. Abdelbacki). https://doi.org/10.1016/j.bbrc.2017.10.008 0006-291X/© 2017 Elsevier Inc. All rights reserved.
Recently, extremely low electromagnetic waves of very low field intensity which resonates with bioelectric signals generated during a particular metabolic activity are used to control the cellular activity of microorganisms. These experiments succeeded to control the growth of Ehrlich tumors in mice [7,8], fungi [9] and bacteria [10,11]. Therefore, the aim of the present study was to find out the resonance frequency of the electromagnetic waves that inhibit the activity of A. tumefaciens and overlap its ability to make division, as well as to determine the changes that may occur at the molecular level as a result of exposure to electromagnetic fields (ELF-EMFs). 2. Materials and methods 2.1. Microorganism growth conditions The strain of A. tumefaciens (ATCC 19358) used in this study was obtained from Microbiological Resources Center (Cairo MIRCEN), Faculty of Agriculture, Ain Shams University, Egypt. Subculture broth media was prepared by inoculating a test tube containing 5 ml sterile nutrient broth/pH 7.1 (Biolife, Milan, Italy) with two
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single colonies of bacteria, then incubated at 28 C for 48 h.
was considered.
2.2. Bacterial reproduction
2.8. Dielectric measurements
The Bacterial growth of A. tumefaciens was determined by measuring the absorbance at 600 nm (A600) every 1 h using spectrophotometer model (6405 UV/Vis) manufactured by Jenway in UK.The absorbance of bacterial suspension then plotted as a function of incubation time.
Bacterial suspensions were subjected to centrifugation at 14,000 rpm at 4 C for 15 min, the pellets were taken and resuspended in a 1 ml volume of sterile deionized water, then the tubes were centrifuged and the pellets were washed with deionized water twice more, finally the pellets were re-suspended in sterile deionized water. A fixed concentration of bacterial cells (107 cfu/ml) was used. The dielectric measurements were done for all samples in the range frequency (f) 0.1e4 MHz using a Loss Factor Meter (type HIOKI 3532 LCR Hi TESTER, version 1.02, Japan), using a sample cell (PW 9510/60; Philips, Weisshausstrasse, Aachen, Germany). The sample cell has two squared platinum black electrodes of area (A) 0.64 cm2 each with an inter-electrode distance (d) of 1 cm. During measurements, the sample between the electrodes was kept at a constant temperature of 24 ± 0.1 C. The capacitance (C) of the samples was measured at each frequency and the resistance was recorded. Each run was taken three times and the average was calculated. The relative permittivity ὲ, loss tangent 00 “tan d”, dielectric loss ε , conductivity s and relaxation time (t) of the samples were calculated for each frequency using the following relation:
2.3. Count-absorbance calibration curve A standard calibration curve was constructed between the absorbance of different concentrations of bacterial suspension and the number of colony forming units (CFU) per ml which determined by plate counting technique [12]. 2.4. ELF EM exposure system Samples of A. tumefaciens suspension were exposed to different extremely low frequencies of square amplitude modulating waves (QAMW). The modulating waveform was square and the carrier frequency was 10 MHz sine wave. The wave carrier was generated by a wave generator model AFG 310 manufactured by Sony Tektronics, Japan, and the modulating wave was generated by synthesized arbitrary generator type TTi TGA1230 manufactured by Thurlby Thandar Instruments Ltd, England. The amplitude of the wave carrier was 10 Vpp and the modulating depth was ±2 V. Different groups of A. tumefaciens were exposed to different frequencies of the QAMW through two parallel cupper electrodes of dimensions 5 5 cm2 and separation distance 1 cm with constant field strength of 200 V/m.
ε0 ¼ Cd=ε0 A tand ¼
(1)
. 00 1 pfRC ¼ ε ε0 2
(2)
00
s ¼ 2pf,ε ε0
(3)
1 pf c 2
(4)
2.5. Determination of resonance frequency of growth inhibition
T¼
The A. tumefaciens suspension was divided into thirteen groups (each group contains three samples of A. tumefaciens suspension), one group kept as control and the other twelve groups exposed to different frequencies of QAMW in the range from 0.1 to 1.3 Hz in steps of 0.1 Hz for a period of 60 min. At the end of the exposure period, the control and exposed groups were incubated at 28 C and every 1 h the incubations were interrupted for absorbance measurements at 600 nm in order to determine the resonance frequency of growth inhibition.
Where, εo is the permittivity of free space, fc is the frequency at the midpoint of the dielectric dispersion curve and R is the resistance of the specimen in Ohm.
2.6. Determination of the optimum exposure time Eight groups of A. tumefaciens suspension were prepared; one kept as control and the other groups were exposed to QAMW at the inhibiting resonance frequency for periods (60, 90, 120 and up to 150 min). At the end of the exposure time; the samples were incubated at 28 C. Every 1-h the samples were shaken and the absorbance were measured then the corresponding number of cells was calculated from the calibration curve. 2.7. Antibiotic sensitivity test A. tumefaciens was subjected to susceptibility test using the following antimicrobial agents: Amikacin 30 mg (AK), Gatifloxacin 5 mg (GAT), Ciprofloxacin 5 mg (CIP), Carbenicillin 100 mg (PY), Chloramphenicol 30 mg (C), Gentamicin 10 mg (CN), Cefaclor 30 mg (CEC), and Rifampin 5 mg (RA). Antimicrobial sensitivity test was carried out and performed by the procedure outlined by [13]. The sensitivity test was carried three times for each of the treated samples and the control, then the average value for the readings
2.9. DNA analysis The DNA extraction was made from 50 mg of fresh culture of A. tumefaciens according to the method modified and developed by [14]. The quality and quantity of the DNA was measured by means of both agarose gel electrophoresis and spectrophotometer. Code and nucleotide sequence of tested primers used in the random amplified polymorphic DNA (RAPD) reactions is shown in Table 1. RAPD reactions were performed according to [15]. RAPD reactions were conducted in a Techne TC-412 thermocycler (Barloworld Scientific Ltd, United Kingdom). Mid-range DNA Ladder (100e3000 bp) (Jena Bioscience, place, Germany) was used and gel documentation system (AAB Advanced American Biotechnology 1166 E. Valencia Dr. Unit 6 C, Fullerton, CA 92631) and The similarity level was determined by un-weighted pair group method based on arithmetic mean (UPGMA).
Table 1 RAPD primer: names and sequences. Primer name
Primer sequence
OPA-02 OPA-10 OPA-13 OPK-08 OPK-10
50 -TGCCGAGCTG-30 50 -GTGATCGCAG-30 50 -CAGCACCCAC-30 50 -GAACACTGGG-30 50 -GTGCAACGTG-30
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2.10. Transmission electron microscope (TEM) examination The morphological changes of A. tumefaciens due to the exposing to QAMW at resonance frequency for the most effective time have been determined using TEM (JEM-1400; JEOL Ltd., Akishima, Tokyo, Japan) presented at Faculty of Agriculture Research Park-Cairo University. The bacterial samples passed through some processing according to [16]. Images were captured by CCD camera model AMT, optronics camera with 1632 1632 pixel format as side mount configuration. 2.11. The pathogenicity test of A. tumefaciens strain To determine the pathogenicity of A. tumefaciens strains before and after exposing to QAMW; 10 ml of bacterial suspension has a concentration of 109 CFU/ml was prepared and divided equally into two groups, one group kept as control and the other one exposed to QAMW at resonance frequency of growth inhibition for the most effective time. Healthy tomato plants were planted and divided into three groups, each group contained five plants. A pin prick was then made into the stems of all plants and three drops of sterile deionized water were placed at the wound sites in the stem of the first group. The second group was infected with non-exposed bacterial suspension and the last group was infected with exposed bacterial suspension. Symptoms were then followed-up after inoculation and the photographs were taken 4e6 weeks later. 2.12. Statistical analysis The statistical analysis was performed using student's t-test and ANOVA analyses, with a minimal confidence level of 0.05 for statistical significance. Each experimental study was performed at least three times with a minimum of three samples per termination point.
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1 h (P < 0.01). From the standard calibration curve, the number of CFU/ml at stationary phase for the control and group exposed to 1.0 Hz QAMW for 1hr was calculated as shown in Table 2. It is clear from the data that the population intensity of the exposed samples had been reduced as compared to that of the control with highly significant inhibition (P < 0.01).
3.2. Effects of exposure time The growth inhibition percentages of A. tumefaciens after the exposure to 1.0 Hz QAMW for different exposure periods at 12 h of incubation are shown in Table 3. The results showed that 90 min exposure gave the maximum growth inhibition at the resonance frequency (49.2%).
3.3. antibiotic susceptibility test The antibiotic susceptibility test results for control and exposed to 1.0 Hz QAMW samples are given in Table 4. It is clear that exposure to QAMW at 1.0 Hz for 90 min (the most effective time) gave significant increase in the sensitivity of A. tumefaciens to all antimicrobial agents used.
3.4. Effects on the molecular structure 3.4.1. Dielectric relaxation Table 5, represented the values of the relaxation time (t), the dielectric increment (Dέ) and the electric conductivity (s) for control and exposed group. The data obtained indicated pronounced decrease in the relaxation time, dielectric increment and conductivity for the exposed sample compared with the control ones.
3. Results 3.1. Growth curve characteristics of A. tumefaciens Fig. 1, showed the differences in absorbance between bacterial groups exposed to QAMW in the range 0.0 Hz up to 1.3 Hz for 1 h and control group at 12 h incubation time as a function of the applied frequency. The data indicated a highly significant growth inhibition occurred after exposure to 1.0 Hz QAMW for
3.4.2. DNA analysis Three out of five screened primers provided informative RAPD pattern for all isolates. Fig. 2a, indicates the appearance of new band in the exposed samples at 490 bp for primer OPA-10, however, Fig. 2b and c, showed disappearance of 3 bands in the exposed samples; at 750, 420 bp, for OPA-13 primer and at 900 bp for OPK08 primer. There is no difference in RAPD patterns for both control and exposed samples appeared for primers OPA-02 and OPK-10.
Fig. 1. The change in absorbance post 12 h of incubation with respect to control as a function of frequency.
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M.A. Fadel et al. / Biochemical and Biophysical Research Communications 494 (2017) 365e371 Table 2 The concentration of A. tumefaciens in (cfu/ml) before and after exposure to 1.0 Hz QAMW for 1hr with its inhibition percentage difference. Sample
N at the stationary phase x 108 (cfu/ml)
Growth Inhibition Percentage G%
Control 1.0 Hz QAMW
(14.6 ± 0.3) (11.2 ± 0.21)
0% (23.3%)**
NS
Not Significant. Significant (P < 0.05). Highly Significant (P < 0.01). *** Very High Significant (P < 0.001). *
**
Table 3 The growth inhibition percentages of A. tumefaciens exposed to 1.0 Hz QAMW for different exposure periods at 12 h of incubation. Exposure time (min) Growth inhibition percentage (G %)
30 (10.7%)*
45 (14.5%)*
60 (23.3%)**
75 (32.2%)**
90 (49.2%)**
120 (30.4%)**
150 (29%)**
NS
Not Significant. Significant (P < 0.05). Highly Significant (P < 0.01). *** Very High Significant (P < 0.001). *
**
Table 4 The mean of the inhibition zones diameter of different antimicrobial agents for control and sample exposed to 1.0 Hz QAMW for 90 min. Frequency
Antibiotic Mean inhibition zone diameter (mm) C
CN
AK
Inhibitors for protein synthesis Control 1.0 Hz QAMW
(26.5 ± 0.2) (35 ± 0.3)**
(23 ± 0.26) (30 ± 0.2)**
(23.5 ± 0.4) (33 ± 0.32)***
GAT
CIP
PY
CEC
RA
Inhibitors for cell wall synthesis
Inhibitors for DNA
Inhibitors for RNA synthesis
(25 ± 0.3) (34 ± 0.2)***
No effect (11 ± 0.13)**
(12 ± 0.36) (25 ± 0.41)***
(24 ± 0.12) (32 ± 0.24)**
No effect (13 ± 0.42)**
NS
Not Significant. Significant (P < 0.05). Highly Significant (P < 0.01). *** Very High Significant (P < 0.001). *
**
Table 5 The relaxation time (t), dielectric increment (Dέ) and conductivity (s) of control and exposed to 1.0 Hz QAMW for 90 min samples. Sample/Dielectric parameter
Relaxation time (t) (107sec)
Dὲ¼(ὲ0 - ὲ∞)
Conductivity (s) at 4 MHz (x107 S/m)
Control (unexposed) 1.0 Hz QAMW
(0.49 ± 0.24) (0.31 ± 0.72)*
(246 ± 9.21) (166 ± 2.75)***
(56 ± 3.57) (43 ± 1.53)**
NS
Not Significant. Significant (P < 0.05). ** Highly Significant (P < 0.01). *** Very High Significant (P < 0.001). *
Fig. 2. Electrophoretic RAPD patterns (a) OPA-10 primer, (b) OPA-13 primer and (c) OPK-08 primer. M ¼ DNA Ladder (DNA Marker). C ¼ DNA of the control sample. E ¼ DNA of the treated sample exposed to 1.0 Hz QAMW for 90 min.
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3.5. Bacterial images by TEM
4. Discussion
Fig. 3a and b showed TEM images for the control A. tumefaciens cells. The majority of the cells have a well preserved cell envelope that consists of a smooth, evenly stained plasma membrane, a semi translucent periplasmic gel and a symmetrically stained, taut outer membrane. Additionally, the cells look remarkably robust and their chromosome is spread throughout the entire cytoplasm over the binary fission which confirming an active metabolic processes. Images of exposed cells to 1.0 Hz QAMW are shown in Fig. 3c and d; where, fragmentation of DNA, disruption and disintegration of cell wall and cytoplasmic membrane, an extrusion of cytoplasmic contents from cell wall and abnormal septation were observed.
The present work introduced a new method for controlling the crown gall disease via inhibition of A. tumefaciens growth using extremely low frequency electromagnetic fields (ELF-EMF) at resonance frequency of growth inhibition. The present findings indicated that the exposure to 1.0 Hz QAMW for 1 h caused highly significant growth inhibition for A. tumefaciens (Fig. 1 and Table 2) and confirming that it is the resonance frequency of growth inhibition for this bacteria. The inhibition effect of the ELF-EMF is due to the interference of this field according to its frequency with the bioelectric signals generated from physiological functions of bacterial cells. The results of these interference reactions depend on the mode of interference pattern which may lead to inhibition (destructive mode) or enhancement (constructive mode) to the running physiological process [17]. The exposure of bacteria to ELFEMF is associated with many biological effects include; changes on cellular division and cell morphotype [18], DNA and gene expression alterations [19,20] protein synthesis [21] and transport of ions by cell membranes [22]. To illuminate the induced changes that may be occurred in the cellular membrane structure as a result of exposure to QAMW at resonance frequency two experiments were done, a) The dielectric properties and b) Antibiotic sensitivity test. The dielectric relaxation measurements as shown in Table 5, indicated pronounced decrease in the average values of the dielectric increment (Dέ), the relaxation time (t) and the electrical conductivity (s) for exposed bacterial samples. Of note, the electrical conductivity and relaxation time are directly related to the electric dipole moment of the macromolecule, which in turn dependent on
3.6. In vivo results As shown in Fig. 4aee, the symptoms of crown gall disease appeared on the crown and stems of infected plants 4e6 weeks after inoculation by unexposed bacteria. The symptoms include; sudden wilting of the plant, large tumors-like swellings (galls), droopy appearance of branches and the branches gradually turned bronze and die (Fig. 4b). It was found that the tumors were larger in size and more in number, and all pathological symptoms were very aggressive in tomato plants treated with non-exposed bacteria (Fig. 4d) comparing with those treated with exposed bacteria (Fig. 4c and e), which seemed to be quite similar to the healthy plants in Fig. 4a.
Fig. 3. TEM images of (a) Normal A. tumefaciens. (b) Normal A. tumefaciens cells during the binary fission. (c) Exposed A. tumefaciens cells showing abnormal septation (arrow 1), pores were formed inside the cell and in cell membrane and disorganization of cell contents during the cell binary fission (arrows 2, 3 and 4). (d) Exposed A. tumefaciens showed the dissolution of the cell wall and an extrusion of cytoplasmic contents from cell wall.
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Fig. 4. Symptoms on the plants 4e6 weeks after inoculation: (a) Healthy plants treated with deionized water, (b) plants treated with non-exposed bacteria, (c) plants treated with exposed bacteria, (d) plants treated with non-exposed bacteria and (e) plants treated with exposed bacteria to 1.0 Hz QAMW for 90 min.
its size and charge. This decrease in the dielectric parameters after exposure to ELF-EMF may be due to that the exposure of bacterial cells to these fields is associated with heat shock response (HSR) [23,24]. This HSR causes decrease in dielectric permittivity and cell polarizability [25]; and hence affect the cellular charges distribution. In view of the potential role of ELF-EMF in modulating charge movements on the membrane, it has been verified that ELF-EMF can affect membrane functions not only by local effects on ligand binding or ion fluxes but also by altering the distribution and/or the aggregation of the intra membrane proteins [26]. This analysis is supported by the data obtained from antibiotic sensitivity test results using different antimicrobial agents of different interaction mechanisms with the bacteria. As shown in Table 4, the highly significant increase in the sensitivity of A. tumefaciens after the exposure to the antibiotics AK, C and CN that are inhibitors for protein synthesis may be due to an enhancement in the penetration of aminolglycosides into bacteria which are cationic antibiotics that bind to anionic components of the bacterial cell membrane in a reversible and concentration-dependent manner [27]. In addition, the susceptibility increase of the microorganism to cell wall synthesis inhibitors (PY and CEC) after exposure may be due to the effect of electromagnetic waves on the enzymatic activity involved in the bacterial cell wall synthesis. This analysis are supported by TEM images, where, the microbial cells exposed to 1.0 Hz QAMW showed disruption and disintegration of cell wall and cytoplasmic membrane which permitted an extrusion of cytoplasmic contents. All these results support each other and can lead to the conclusion that exposure of A. tumefaciens to 1.0 Hz QAMW caused changes in the properties of cell structure which may affect the cellular activity of the microorganism and cell to cell communication. The appearance of new band in the amplified DNA in the exposed sample prove that the DNA sequences have been changed under the effect of QAMW in a way that the primer used find a new binding sequence which not present in the control sample. Also the absence of bands indicated that the genetic sequences of bacterial DNA were modified due to the exposure to 1.0 Hz QAMW. These DNA
mutations and genetic alterations resulted in changes in the translated proteins and enzymes involved in bacterial growth process and divisions [28,29]. These explain the highly significant increase in the sensitivity of A. tumefaciens after the exposure to the antibiotics CIP and GAT which are inhibitors for DNA after the exposure to 1.0 Hz QAMW. The in-vivo results showed that the symptoms of crown gall were more aggressive in plants treated with non-exposed bacteria comparing with those treated with exposed bacteria, which seemed to be quite similar to the healthy plants. The crown gall disease was appeared with very slight symptoms in the plants treated by exposed bacteria and almost all plants treated by non-exposed bacteria were hit by aggressive symptoms of crown gall disease. Based on these finding, it could be suggested that the exposure to ELF-EMF at the inhibiting resonance frequency to cellular growth affect the physiological behavior and pathogenic genes of A. tumefaciens, which in turn affect the pathogenicity of the bacteria as confirmed by DNA and in-vivo results. All of these changes can cause the weakening of the exposed bacterial cells, which in turn gave a chance to the defense mechanism of plant to overcome the pathogen and prevent the disease. We concluded from the present findings that the exposure of A. tumefaciens to QAMW at the inhibiting resonance frequency is a new promising technique as an aid to avoid the use of traditional disease control techniques or the use of antibiotics and soil fumigants to control the crown gall disease. Declaration of interest This work is a part of the Ph.D. thesis of Shaimaa Abd El-Raof Mohamed submitted to the Biophysics Department, Faculty of Science, Cairo University. No external funding was given and/or available to run this work. Conflicts of interest None.
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References [1] M.D. Chilton, M.H. Drummond, D.J. Merio, et al., Stable incorporation of plasmid DNA into higher plant cells: the molecular basis of crown gall tumorigenesis, Cell 11 (1977) 263e271. [2] S. Murakoshi, M. Takahashi, Trials of some control of tomato wilt caused by Pseudomonas solanacearum, Bull. Kanagawa Hortic. Exp. Stn. 31 (1984) 50e56. [3] G. Hartman, J. Elphinstone, Advances in the control of Pseudomonas solanacearum race 1 in major food crops, in: A. Hayward, G. Hartman (Eds.), Bacterial Wilt: the Disease and its Causative Agent, Pseudomonas Solanacearum, CAB International, Wallingford, UK, 1994, pp. 157e178. [4] M. Lopez, E. Biosca, Potato bacterial wilt management: new prospects for an old problem, in: C. Allen, P. Prior, A.C. Hayward (Eds.), Bacterial Wilt Disease and the Ralstonia Species Complex, APS Press, St. Paul, Minnesota, USA, 2004, pp. 205e224. [5] Z. Liang, Z. Cheng, G. Mittal, Inactivation of spoilage microorganisms in apple cider using a continuous flow pulsed electric field system, LWT 39 (2006) 350e356. [6] I. Ayse, A. Burak, A. Zafer, A. Dilek, A. Nilufer, S. Tangul, Effect of extremely low frequency electromagnetic fields on growth rate and morphology of bacteria, Int. J. Radiat. Biol. (2011) 1e8. [7] M.A. Fadel, R. El-Gebaly, A. Aly, A. Sallam, O. Sarhan, H. Eltohamy, Preventing of Ehrlich tumor metastasis in liver, kidney and spleen by electromagnetic field, Int. J. Phys. Sci. 5 (2010) 2057e2065. [8] M.A. Fadel, R. El-Gebaly, A. Hamad, Combination of bacteriolytic therapy with magnetic field for Ehrlich tumour treatment, Gen. Physiol. Biophys. 36 (2017) 259e271. [9] M.A. Fadel, M.A. Ahmed, M.A. El Hag, Control of Sclerotium cepivorum (Allium White Rot) activities by electromagnetic waves at resonance frequency, Aust. J. Basic & Appl. Sci. 3 (2009) 1994e2000. [10] M.A. Fadel, S. Mohamed, A.M. Abdelbacki, A. El-Sharkawy, Inhibition of Salmonella typhi growth using extremely low frequency electromagnetic (ELFEM) waves at resonance frequency, J. Appl. Microbiol. 117 (2014) 358e365. [11] M.A. Fadel, R. Elgebaly, M. Elneklawi, A. Othman, Role of duty cycle on Pseudomonas aeruginosa growth inhibition mechanisms by positive electric pulses, Bio-Med. Mat. Eng 27 (2016) 211e225. [12] R. Atlas, Principles of Microbiology, Mosby- Year book, INC, 1995. [13] National Committee for Clinical Laboratory Standards (Ed.), Performance Standards for Antimicrobial Disk Susceptibility Tests, third ed., National Committee for Clinical Laboratory Standards Villanova, Pa, 1985. [14] S.L. Dellaporta, J. Wood, J.B. Hicks, A plant DNA minipreparation: version II, Plant Mol. Biol. Report. 1 (1983) 19e21.
371
[15] J.G.K. Williams, A.R. Kubelik, K.J. Livak, J.A. Rafalski, S.V. Tingey, DNA polymorphisms amplified by arbitrary primers are useful as genetic markers, Nucleic Acids Res. 18 (1990) 6531e6535. [16] M. Demicheli, A. Goes, A. Ribeiro, Ultrastructural changes in Paracoccidioides brasiliensis yeast cells attenuated by gamma irradiation, J. Compil. 50 (2007) 397e402. [17] M.A. Fadel, H.E. Reem, A.A. Amany, F.I. Fakhry, Control of Ehrlich tumor growth in mice by electromagnetic wave at resonance frequency, in vivo, Electromagn. Biol Med 24 (2005) 9e12. [18] L. Cellini, R. Grande, E. Di Campli, et al., Bacterial response to the exposure of 50 Hz electromagnetic fields, Bioelectromagnetics 29 (2008) 302e311. [19] J. Galvanoskis, J. Sandblom, Periodic forcing of intracellular calcium oscillators Theoretical studies of the effects of low frequency fields on the magnitude of oscillations, Bioelectrochem Bioenerg. 2 (46) (1999) 161e174. [20] A. El May, S. Snoussi, N. Ben Miloud, et al., Effects of static magnetic field on cell growth, viability, and differential gene expression in Salmonella, Foodborne Pathog. Dis. 6 (2009) 547e552. [21] E.M. Goodman, B. Greenebaum, M.T. Marron, Altered protein synthesis in a cell-free system exposed to a sinusoidal magnetic field, Biochim. Biophys. Acta 1202 (1993) 107e112. [22] G. D'Inzeo, S. Pisa, L. Tarricone, Ionic channel gating under electromagnetic exposure: a stochastic model, Bioelectrochem Bioenerg. 29 (3) (1993) 289e304. [23] O. Smith, Cells, stress and EMFs, Nat. Med. 2 (1996) 23e24. [24] B. Del Re, P. Marcantonio, F. Bersani, G. Giorgi, Extremely low frequency magnetic field exposure affects DnaK and GroEL expression in E. coli cells with impaired heat shock response, Gen. Physiol. Biophys. 28 (2009) 420e424. [25] Z. Boris, Z. Zhihui, N. Aphakorn, J. Ronald, Rapid microbiological testing: monitoring the development of bacterial stress, PLoS One 5 (10) (2010) 1e8 e13374. [26] A. Marchionni, M. Paffi, P. Pellegrino, Comparison between low-level 50 Hz and 900MHz electromagnetic stimulation on single channel ionic currents and on firing frequency in dorsal root ganglion isolated neurons, Biochimica Biophysica Acta 1758 (5) (2006) 597e605. [27] H.W. Taber, J.P. Mueller, P.F. Miller, A.S. Arrow, Bacterial uptake of aminoglycoside antibiotics, Microbiol. Rev. 51 (1987) 439e457. [28] J. Van der Wolf, J. Van Beckhoven, E. De Haan, G. Van den Bovenkamp, G. Leone, Specific detection of Ralstonia solanacearum 16S rRNA sequences by AmpliDet RNA, Eur. J. Plant Pathology 110 (2004) 25e33. [29] Y. Lee, S. Fan, L. Chiu, K. Hsia, Isolation of an insertion sequence from Ralstonia solanacearum race 1 and its potential use for strain characterization and detection, Appl. Environ. Microbiol. 67 (2001) 3943e3950.