الفهرس 
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Abstract
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CHAPTER ONE
INTRODUCTION
Ralstonia solanacearum (race 3 biovar 2), the causative agent of potato brown rot disease (bacterial wilt), is an economically important disease in tropical, subtropical and temperate regions of the world [1].
Potatoes are Egypt’s largest horticultural export crop. Yet, it is considered one of the most important hosts of Ralstonia solanacerum which causes qualitative and quantitative yield losses. The existence of this disease in soil hinders the cultivation of potato and the production of seed tubers or exportation. R. solanacerum is a quarantine organism and so, there can be large costs to disease testing and administration of seed production to control the disease. The total value of Egyptian potato exports fell from a peak value of US$ 102.12 million in 1995 to US$ 7.7 million in 2000 mainly due to quarantine restrictions on the potato brown rot imposed by the European Union (EU) which used to account for about 70-90% of Egyptian potato exports. Therefore, The Central Administration for Plant Quarantine (CAPQ) of Ministry of Agriculture, Egypt, has recently set up a new Directorate for Internal Potato Quarantine in order to delimit pest free areas (PFAs), i.e. areas in which R. solanacearum the pathogen of brown rot has not been known yet [2].
The main concerned dangers of this pathogen is its survive in soil for extended periods without a host plant. It enters roots through wounds, which may be caused by insects, nematodes, and cultivation. High temperature and high soil moisture favor disease development. Control of such disease requires integrated cultural practices and chemical sprays with copper compounds, but its available measures are not effective and one of the major limitations of using chemical control agents is the development of resistance in bacteria [3]. On the same regard the use of agrochemicals is becoming less favorable because of environmental pollution and detrimental effect on a variety of non–target organisms [4].
Several trials have been carried out all over the world to control the disease but without much success. No promising control of brown rot was achieved using antibiotics [5], soil fumigants [6], chemical control [7] or breeding of resistant varieties [8,9]. Also, biocontrol agents were found to be relatively ineffective in the control of R. solanacearum populations under natural conditions [10,11]. The failure probably results from the inability of the introduced biocontrol agent to establish itself and to produce the desired compound under the stress of competition by the native microbial community.
Over the last few years, efforts had been devoted to control bacterial growth through exposure to Pulsed electric fields (PEF) which represents one of the most promising minimal processing technologies, and have been proven to inactivate microorganisms under non-thermal conditions where it has been shown to be effective in eliminating pathogens and spoilage organisms and capable of producing safe fresh-like
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food [12]. So several studies have been focused recently toward the effects of PEF on inactivation of the pathogenic bacteria viability.
Dutreux et al. (2000) [13] used the PEF (10 pulses at an intensity of 41 kV/ cm and at 37°C) in the inactivation of Escherichia coli and Listeria innocua suspended in milk and phosphate buffer. The results showed that the PEF treatment of both Escherichia coli and Listeria innocua attached to polystyrene beads gave higher inactivation rates than pulsed electric field treatment of non-attached bacteria. Simultaneously, through the examination by transmission and scanning electron microscopy, they observed changes in the cytoplasm, the cell surface appeared rough and the cells outer membranes were partially destroyed allowing leaking of cell cytoplasm.
Grzesiuk et al. (2001) [14] stated that, intestinal bacteria, particularly those adhering to intestinal epithelial cells, were exposed to electric field are similar to that generated by the muscular activity of the small intestine. The duodenal signal obtained via serosal electrodes from a healthy calf was recorded and then applied via platinum electrodes to Escherichia coli cultures. The culture tubes were placed within a Faraday shield, incubated at 37°C with shaking and exposed to the field for 5 or 8 h. The results showed that the growth of the exposed Escherichia coli was significantly altered compared to those of controls. The authors suggested that the myoelectrical activity of the duodenum, through action on cell membrane, could affect cell division of intestinal bacteria.
Cserhalmi et al. (2002) [15] examined the inactivation effect of PEF treatment (Applying of 8.3 pulses for 2 μs each at 25 kV/cm) on Saccharomyces cerevisiae and Bacillus cereus cells and spores. resulted in 3.3 log cycles reduction of the Saccharomyces cerevisiae cells suspended in apple juice and only 1.3 log cycles reduction of Bacillus cereus cells suspended in NaCl. Also, by increasing the pulse number from 8.3 to (10.4 at 20 kV/cm electric field intensity), the inactivation of Saccharomyces cerevisiae increased to about 3.9 log cycles. However, at 20 kV/cm electric field intensity for pulse number above 4 practically did not further decrease in cell count of Bacillus cereus cells which confirmed that Saccharomyces cerevisiae cells were more sensitive to the PEF than Bacillus cereus cells and it was practically ineffective to Bacillus cereus spores.
Rodrigo et al. (2003) [16] studied the kinetics of Lactobacillus plantarum inactivation by PEFs in two different growth stages (exponential and stationary). Application of electric field intensity and treatment time varied from 20 to 28 kV/cm and 30 to 240 μs, respectively. Results showed that cells in the exponential growth stage were more sensitive to PEF treatment than those in the stationary stage.
Amiali et al. (2004) [17] investigated inactivation characteristics of Escherichia coli in three different mediums of liquid egg products at low temperature using square waveform pulsed electric fields. Dialyzed liquid egg products, namely whole egg, egg white and egg yolk, were exposed to an electric field of 15 kV/ cm, up to 500 pulses and a pulse frequency of 1Hz at a low temperature of 0°C . They found that about 1, 3 and 3.5 log reductions were obtained for the dialyzed egg white, egg yolk and whole egg products, respectively. The results indicated that microbial inactivation rate increased with increasing number of pulses, especially for the egg yolk and whole egg products.
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Aronsson et al. (2005) [18] examined the inactivation of Escherichia coli, Listeria innocua and Saccharomyces cerevisiae in relation to membrane permeabilization and subsequent leakage of intracellular compounds due to PEF treatment at (30 kV/cm and 4 μs). The results indicated that Saccharomyces cerevisiae was the most PEF sensitive, followed by Escherichia coli and Listeria innocua. Through the determination of propidium iodide (PI) uptake in three strains of bacteria, they found that a higher electrical field strength and a longer pulse duration resulted in a greater numbers of permeated cells for all strains but with large dependence on the type of microorganism, with Listeria innocua was the most resistant, followed by Escherichia coli, and Saccharomyces cerevisiae as the most sensitive to PEF treatment.
Liang et al. (2006) [19] studied the effect of PEF on inactivating naturally occurring microorganisms (yeast and molds) in freshly squeezed apple cider. At field strength of 27–33 kV/cm, 200 pulse/s and 10 liter/h flow rate, there was a 1.12 log cycles reduction in microbial counts. However, when samples were treated at 3 liter/h flow rate with the same number of pulses and field strength, a 3.1 log cycles reduction was achieved. It was revealed that the microbial count decreased with an increase of applied pulses and field strength, and a decrease of flow rate. Also, the PEF treatment in the presence of a mixture from nisin and lysozyme, there was an increase in the microbial count from 1.12 to 1.78 log reductions. They concluded that the greater viability loss of cells due to pulsed electric field treatment in the presence of antimicrobials was synergistic.
Amiali et al. (2007) [20] investigated the inactivation effect of pulsed electric field (PEF) on Escherichia coli and Salmonella enteritidis in liquid egg yolk as a function of the field strength and temperature. Exposure to PEF of strength 20 kV /cm for 210 μs at temperature of 20°C, resulted in 1.4 and 0.5 log cycles reduction in microbial counts for both Escherichia coli and Salmonella enteritidis, respectively. However, when the two pathogens were exposed to 30 kV/cm for the same 210 μs treatment time at 40°C, 4.9 and 4.8 log reduction were observed in Escherichia coli and Salmonella enteritidis, respectively. It was observed that Salmonella enteritidis was more resistant to PEF inactivation than Escherichia coli at lower processing temperatures. Moreover, the increase of the applied electric field intensity and process temperature resulted in increase bacterial inactivation.
Fox et al. (2008) [21] examined the effects of field strength, number of pulses, pulse width and temperature on the inactivation of Lactobacillus plantarum by PEF. The results showed that the microbial count of Lactobacillus plantarum decreased with an increase of applied pulses and field strength. Higher inactivation rate of 1.6 log reduction was achieved by exposing the bacterial cells to electric field strength of 38 kV/cm and 24 pulses. Changing the pulse width at constant field strength and pulse number did not show any change in microbial inactivation. Furthermore, treatment at higher temperatures showed higher inactivation rates which mean that both PEF and processing temperature had synergistic effect on the inactivation of microorganisms.
Kambiz et al. (2009) [12] reported that recently several studies have been focused to the use of PEF in the food industry which was considering largely a non-thermal process that is able to inactivate microorganisms and enzymes to some degree in liquid food such as milk and fruit juice which is reported to have minimum adverse effects on the
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sensory attributes of these products. The PEF process was considered to be energy efficient since the microbial inactivation was achieved at ambient or moderately elevated temperatures by the application of short bursts of high intensity electric fields to liquid food flowing between two electrodes.
Fadel et al. (2009) [22] exposed Sclerotium cepivorum fungi inoculated on potato dextrose agar (PDA) medium to square amplitude modulated waves (SAMW) from generators with constant carrier frequency of 10 MHz , amplitude of 20 Vpp and modulating depth + 2V. They studied the effects at different modulating frequencies on Sclerotium cepivorum production and observed that there was no Sclerotium when the samples were irradiated at a frequency of 20 Hz SAMW for a single exposure of two hours. The scan step was done at intervals of 0.1 Hz. The data confirmed that there was a resonance frequency only at 20 Hz at whish no Sclerotium were produced. The results indicated the appearance of a new band in the DNA as a result of exposure to 20 Hz whish could be considered as a marker for genetic alterations in the DNA of the exposed microorganism.
Ruiz et al. (2010) [23] studied the effects of extremely-low frequency magnetic field exposure on the growth, cell cycle, survival and DNA damage of mutant Saccharomyces cerevisiae strains deficient in DNA strand breaks repair (hdf1, rad52 and rad52 hdf1). The results showed that exposure of all mutant yeast strains to magnetic field of 2.45 mT, sinusoidal 50 Hz for 96 h induced alterations in the growth and survival of Saccharomyces cerevisiae strains deficient in DNA strand breaks repair. However the magnetic field treatment did not induce alterations in the cell cycle and did not cause DNA damage.
Jie et al. (2011) [24] showed that PEF technology was used for cell disruption prior to extraction of intracellular lipids. Severe cell disruption was evident after PEF treatment, especially with treatment intensity (TI) > 35 kWh/m3. However, the forces associated with the PEF caused significant damage to the plasma membrane, cell wall, and thylakoid membrane, and it even led to complete disruption of some cells into fragments, which resulted in biomass loss. Treatment by PEF enhanced the potential for the low-toxicity solvent isopropanol to access lipid molecules during subsequent solvent extraction, leading to lower usage of isopropanol for the same extraction efficiency. Thus, PEF shows promise for lowering the costs and environmental effects of the lipid-extraction step.
Ayse et al. (2011) [25] determined the effect of extremely low frequency of electromagnetic fields (ELF-EMF) (50 Hz, 0.5 mT) on the growth rate of Six bacterial strains of Gram-positive and Gram-negative bacteria and determine morphological changes that might have been caused by ELF-EMF. A decrease in growth rate with respect to control samples was observed for all strains during ELF-EMF application and the decrease in growth-rate continued when exposed bacteria were cultured without field application, also significant ultrastructural changes were observed in all bacterial strains, which were seen to resemble the alterations caused by cationic peptides. They reported that ELF-EMF induces a decrease in growth rate and morphological changes for both gram-negative and gram-positive bacteria.
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On the same regard, Fadel et al. (2012) [26] investigated the frequency of the magnetic impulses that interfere with the bioelectric signals generated during salmonella typhimurium (STm) cellular division. Then the experiment was expanded to in vivo study for the obtained data in which rats were infected with STm and then whole body were exposed to square magnetic pulses (SMP) that caused inhibition to the microbial cellular growth. Another group of animals was infected by previously inhibited bacteria with SMP then the histological and molecular structures of the liver were investigated for all the animal groups. Dielectric relaxation studies for the liver in the frequency range 100 KHz – 4.5 MHz was used to determine molecular structure changes. The results indicated a highly significant inhibition of cellular growth for STm in addition to pronounced changes in the cellular morphology after the exposure of the microorganism to the resonance frequency of 0.8 Hz SMP for 75 minutes.
Varsik et al. (2013) [27] studied the frequency-dependent effects of ELF EMF on Escherichia coli K-12 growth. To check this effects, the frequency-dependent effects (2, 4, 6, 8, 10 Hz, B = 0.4 mT, 30 minute) of ELF-EMF on the bacterial growth were studied in both cases where the microbes were in the culture media during the exposure and where culture media was preliminarily exposed to the ELF-EMF before the addition of bacteria. It has been shown that EMF at 4 Hz exposure has pronounced stimulation while at 8 Hz it has inhibited cell proliferation.
Fadel et al. (2013) [28] studied the effect of positive square pulsed electric fields at different frequencies in the range 0.1-50 Hz, exposure periods on the growth characteristics of P. aeruginosa. Furthermore, studied the effect of exposure on bacterial antibiotic susceptibility and molecular and morphological cellular structure. Results indicated that exposure to PEF can inhibit bacterial growth at particular resonance frequencies 0.7 Hz and 0.5 Hz and significant increase in antibiotic susceptibility to protein and cell wall inhibitors. Also, results of DNA, dielectric relaxation and TEM indicated molecular and morphological changes.
Hagar (2011) [29] indicated that ELF-EMF had induced morphological alterations and significant ultra structural changes were observed on the bacterial cells of R. Solanacearum (brown rot disease) after exposure to 1.0 Hz either square amplitude modulated wave (SAMW) or pulsed magnetic field (PMF) for 1 h. In addition, the DNA analysis indicated the presence of genetic alterations in the DNA and the samples were treated with 1.0 Hz PMF showed pronounced decreases in the values of dielectric dispersion which is an indicator of the decreasing of the value of the dipole moment of the macromolecule (protein) also electrical conductivity as compared with control.
Electric field is one kind of stress, which can affect directly or indirectly the plant exposed to it. Plant species vary in their sensitivity and response to environmental stresses because they have various capabilities for stress perception, signaling and response [30]. The electrical phenomena in plants have attracted researchers since the eighteenth century [31].
Biologically closed electrical circuits operate over large distances in biological tissues. The activation of such electrical circuits can lead to various physiological and biochemical responses [32]. The cells of many biological organs generate electric
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potentials that can result in the flow of electric currents [33]. Electrical impulses may arise as a result of stimulation. Once initiated, these impulses can propagate to adjacent excitable cells. The change in transmembrane potential can create a wave of depolarization which affects the adjoining, resting membranes. Thus, while the plasma membrane was stimulated at any point, the action potential can propagate over the entire length of the cell membrane and along the conductive bundles of tissue with constant amplitude, duration, and speed [34].
Schoenbach et al. (2001) and Gowrishankar et al. (2006) [35, 36] stated that when applying moderate external electric field amplitudes of several kV/cm and pulse rise times in the microsecond range the electric field along the cell was into the membrane and the electric field across the cell interior was negligible. Whereas for steep and high amplitude electric field pulses, i.e. for pulse rise times which were considerably shorter than the charging time of the membrane, the electric field penetrates into the cell interior. The displacement current can charge intracellular membranes and affect cell organelles. There was a broad variety of responses of mammalian cells to nanosecond pulsed electric field (nsPEF) exposition [37] such as the intracellular calcium release [38, 39], phosphatidylserine externalization [40] and DNA damage [41]. A promising application of nsPEFs was the killing of melanoma cells [42].
The majority of the reported studies indicated an increase in germination rate and plant growth on the order of 10-20 % [43] and observed an accelerated germination when exposing tomato seeds to a 4 – 12 kV/cm ac electric field for one. For also, a positive influence on Potato seeds yield was reported, when treating them for 12 minute with a 4 kV/cm electrostatic field prior to sowing [44].
Hanafy et al. (2001) [45] exposed the broad bean seeds to definite exposure periods (1, 3 and 5 days, respectively) to electric field (6 kV/m-50 Hz) and recorded a considerable increase in each of the chlorophyll content and the carbohydrate amount by increasing the exposure periods, and also considerable changes in the elements level were recorded for the exposed seeds.
Federico et al. (2008) [46] studied the effect of the application of PEFs to potato tissue on the diffusion of the fluorescent dye FM1-43 through the cell wall. Potato tissue was subjected to field strengths ranging from 30 to 500 V/cm, with 1 ms rectangular pulse, before application of FM1-43 and microscopic examination. Results showed a slower diffusion of FM1-43 in the electropulsed tissue when compared with that in the non-pulsed tissue, suggesting that the electric field decreased the cell wall permeability. This is a fast response that is already detected within 30 s after the delivery of the electric field.
Ricardo et al. 2009 [47] subjected potato tissue to nominal field strengths (E) ranging from 30 to 500 V/cm, with a single rectangular pulse of 10−5, 10−4, or 10−3 s. The changes on the viscoelastic properties of potato tissue during PEFs were monitored through small amplitude oscillatory dynamic rheological measurements. The elastic (G’) and viscous moduli (G″) were measured every 30 s after the delivery of the pulse and the loss tangent change (tan-δ) was calculated. The results were correlated with measurements of changes on electrical resistance during the delivery of the pulse. Results showed a drastic increase of tan-δ in the first 30 s after the application of the pulse, followed by a decrease 1
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minute after pulsation. This response is strongly influenced by pulsing conditions and is independent of the total permeabilization achieved by the pulse. results showed that PEF causes a rapid change of the viscoelastic properties of the tissue that could be attributed to a partial loss in turgor pressure and a slight increase of stiffness traduced by a negative change of tan-δ after application of certain PEF conditions may also give an indication of events occurring on cell wall structure due to stress responses.
In recent years, several studies have been accomplished by researchers to evaluate the effects of electric and magnetic fields on the viability of plants and biological commodities [48]. Marinkovic et al. (2002) [49] reported a 144.8% increase in potato yield as a result of electric fields treatment and Navid et al. (2013) [50] studied the physiological properties of maize seeds as affected by electric field intensity (2, 4, 9 and 14 KV) and exposure time (15, 45, 80 and 150 s). Results indicated that increasing the electric field intensity to 9 KV/m caused an increasing trend in the mean germination time (MG) value, there was a significant increase in germination rate (GS) with increasing the electric field intensity from 2 to 9 KV/m and increasing the exposing time caused the length of root to be decrease. The highest decrease was belonged to the 80 s exposing time.
The effect of electric field (EF) on soil can be studied from different points of view. The first point concerned of study deals with the effect of electric field on microorganisms in soil, where this non-ionizing radiation was capable of changing every day, the growth, division and biological function of exposed cells [51]. Another point concerned of study deals with the effect of EF on ionic soil composition causing ion migration and ion movement, which in turn adding another indirect effect of EF on soil microorganism. This phenomena (ion migration) was applied in the processes of soil decontamination and removal of pollutants or to prevent its spreading to surrounding subsoil [52], also in the bioremediation of soil and removal of some hazardous wastes [53].
The interaction of electric field to the cell result in many biological changes. The electric field exerts forces on charged particles in electrically conductive surfaces such as the surface of ground and biological cells induce electrical potentials and resultant current flows in the aqueous medium surrounding the living cells [54]. Because the membranes of these cells form a dielectric barrier, only a small fraction of the induced current penetrates the cell surface producing electrochemical alterations in cell membrane. This event in turn send signals across the cell membrane barrier that produce alteration in intracellular biochemical and physiological function [55].
Theoretically, electric pulse energy charged to soil influences physiological function of some soil bacteria and induces a geochemical change of soil conditions for plants and bacterial communities. The diversity of soil bacterial community was not changed by the electric pulse, contrary to expectation [56]. Accordingly, the effect of electric pulse on activation of lettuce growth may be unrelated to soil bacterial communities since bacterial communities inhabiting soil and rhizosphere improve nutritional conditions for plants [57]. Yi et al. (2012) [58] indicated that electric pulse caused an increase in lettuce growth, and improvement in nutritional soil conditions, and varied mass spectrometric patterns.
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from the previous work one may find out that the most related scientific work is the application of PMF on R. Solanacearum [29] but inside the lab zone without applicability outside the lab for large zones. Hence, it seems necessary to find an alternative technique of high feasibility that could be applied in large farm fields and therefore, this work was conducted.
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Aim of The Work
This work was aimed to find out the resonance frequency of positive electric impulses that may inhibit the activity of R. solanacearum and its most effective exposure time. Moreover, was to investigate the changes that may occur at morphological and DNA molecular level as a result of the exposure. Furthermore, the work was expanded in vivo to study the success of its applicability on farm field zones and the best application conditions.