The
cientific
WorldJOURNAL
Research Article
BiologicalEffectsofWeakElectromagneticFieldonHealthyandInfectedLime(
Citrus aurantifolia
)TreeswithPhytoplasma
FatemehAbdollahi,
1
VahidNiknam,
1
FaezehGhanati,
2
FariborsMasroor,
3
andSeyyedNasrNoorbakhsh
3
1
Department of Plant Sciences, School of Biology and Center of Excellence in Phylogeny of Living Organisms, College of Sciences,University of Tehran, Tehran 14155-6455, Iran
2
Department of Plant Science, Faculty of Biological Science, Tarbiat Modares University, Tehran 14115-154, Iran
3
Department of Chemistry, Engineering Research Institute, Sooliran Street, 16 km Tehran-Karaj Old Road, Tehran 13455-754, Iran
Correspondence should be addressed to Vahid Niknam,vniknam@khayam.ut.ac.irReceived 31 October 2011; Accepted 19 December 2011Academic Editor: Mehmet Zulkuf AkdagCopyright © 2012 Fatemeh Abdollahi et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Exposure to electromagnetic fields (EMF) has become an issue of concern for a great many people and is an active area of research.Phytoplasmas, also known as mycoplasma-like organisms, are wall-less prokaryotes that are pathogens of many plant speciesthroughout the world. E
ff
ects of electromagnetic fields on the changes of lipid peroxidation, content of H
2
O
2
, proline, protein, andcarbohydrateswere investigated inleaves oftwo-year-oldtreesoflime(
Citrus aurantifolia
)infected bytheCandidatus
Phytoplasmaaurantifoliae
. The healthy and infected plants were discontinuously exposed to a 10KHz quadratic EMF with maximum power of 9W for 5 days, each5 h, at 25
◦
C. Fresh and dry weight of leaves, content of MDA, proline, and protein increased in both healthy and infected plants under electromagnetic fields, compared with those of the control plants. Electromagnetic fields decreasedhydrogen peroxide and carbohydrates content in both healthy and infected plants compared to those of the controls.
1.Introduction
During the past years considerable evidence has been accu-mulatedwithnoticetothebiologicale
ff
ectsoflow-frequency electromagnetic fields (EMF), such as those bringing frommodern world such as power lines and household electricalwiring [1,2]. The e
ff
ects of electromagnetic fields of muchlower frequency than visible light on plant growth and devel-opment have rarely been studied until relatively recently, andknowledge is still limited. Several studies have been showedthat low-frequency EMFs may influence plant growth anddevelopment [3–5]. Also the international discussion about
the biological e
ff
ects of electromagnetic fields, in which wewereinvolvedinthepast[6],ledustoexaminethepossibility
of using such fields to inhibit phytoplasmas growth on plantssuch as lime. Phytoplasmas are endocellular prokaryoteswithout cell wall associated with more than 600 diseases inat least 300 plant species [7]. Moreover, knowledge aboutphytoplasmas has been limited by inability to isolate themin pure culture.Reactive oxygen substances (ROS), such as singlet oxy-gen, superoxide anion, and hydroxyl radical, are producedby a free radical chain reaction and may contribute to tissuedamage. To mitigate the oxidative damage initiated by ROS, plants have developed a complex antioxidative de-fense system, including production of low-molecular massantioxidants as well as antioxidative enzymes, such as super-oxide dismutase (SOD), catalase (CAT), ascorbate perox-idase (APX), guaiacol peroxidase (GPX), and glutathionereductase (GR) [8]. Moreover, the level of malondialdehyde(MDA), a product of lipid peroxidation, has also been con-sidered an indicator of oxidative damage under magneticfield [9].Lime (family Rutaceae) is one of the most importantand most economic horticulture products in the south partof Iran. Lime is susceptible to a large number of diseasescaused by plant pathogens [10]. Witches’ broom disease of lime (WBDL) associated with “
Candidatus
Phytoplasmaaurantifolia” is one of the most destructive diseases of limein the southern provinces of Iran [11]. Witches’ broom
2 The Scientific World Journaldisease of lime was first observed in the Sultanate of Omanand later was found to be present in United Arab Emirates[12], India [13], and Iran [14].
Studies on physiological relationships between phyto-plasmas and some host plants have been reported [10,15]
but so far none have focused on the responses of phy-toplasmas-infected lime plants to electromagnetic fields.Thus, the objective of the present work was to study somebiochemical aspects related to lipid peroxidation, content of H
2
O
2
, proline, protein, and carbohydrates in phytoplasmas-infected lime plant under electromagnetic fields
.
2.MaterialsandMethods
2.1. Plant Materials and Electromagnetic Treatment.
Limeplants (
Citrus aurantifolia
L. Swingle cv. Keyline) were in-fected with the Witches’ broom disease of lime (WBDL)
Phytoplasma
by graft transmission and were grown in plasticpots (10
×
10cm) under greenhouse condition in Engineer-ing Research Institute, 16km Tehran-Karaj Old Road, Iran.Infected lime plants used for grafting were collected from
Takht
,
Bandar-e-Abbas
, south of Iran and transported to thegreenhouse. Shoots showing typical symptoms were graftedon two-years-old lime plants.Exposure to EMF was performed by a locally designedelectromagnetic wave generator able to generate di
ff
erentwave shapes including sinusoidal, triangular, and quadratic.The system could generate EMF in range of 0.1Hz–10KHzwith a continuous fine control in stable conditions andmaximum consuming power density of 9W. It was consistedof two vertical coils each 28 turns of 0.3mm copper wirerounded around a quadratic frame of 48
×
34cm. One coilwas oriented in vertical plane (XOZ) with pointing vectorin horizontal direction (antiparallel with the gravity), whilethe other one was oriented in horizontal plane (XOY) withpointing vector in vertical direction (perpendicular with thegravity). Impedance of each coil was 8ohm. The calibrationof the system was performed at di
ff
erent frequencies. Forthe present experiment the healthy and infected plants werediscontinuously exposed to a quadratic EMF for 5 days,each 5h, at 25
◦
C. The applied frequency was 10KHz withconsuming power of 8.3. The average electric and magneticstrength were 168
.
5
±
4 (KV/m) and 3400
±
43 (mA/m),respectively [16]. All measurements were conducted either infresh harvested tissue ground immediately after excision orfrom leaves quickly deep frozen in liquid nitrogen and keptat
−
80
◦
C until the assay.
2.2. Plant Water Relations.
=
fresh mass
−
dry mass
fresh mass
×
100
.
(1)
2.3. Protein Content.
For determination of protein content,500mg fresh leaf was homogenized in a chilled (4
◦
C) mortarusing a 50mM Tris-HCl bu
ff
er (pH 7.0) containing 10mMEDTA, 2mM MgSO
4
, 20mM dithiotreitol, 10% (v/v) glyc-erol, and 2% (m/v) polyvinylpyrrolidone. After centrifuga-tionat13000gfor45minat4
◦
C,thesupernatantwasfilteredand then transferred to Eppendorf tubes and the sample kepton ice at 4
◦
C. A portion of eluent was stored at
−
70
◦
C. Totalprotein content was measured by the spectrophotometricmethod of Bradford[18] using bovine serum albumin (BSA)
as the standard.
2.4. Proline Content.
Free proline content was determinedaccording to Bates et al. [19] using L-proline as a standard.High-speed centrifuge (Beckman J2-21M, Palo Alto, USA)and UV-visible spectrophotometer (Shimadzu UV-160,Tokyo, Japan) with 10mm matched quartz cells were usedfor centrifugation of the extracts and determination of theabsorbance, respectively.
2.5. Malondialdehyde Content.
The level of lipid peroxida-tion was measured in terms of thiobarbituric acid reactivesubstances (TBARS), following the method of Heath andPacker [20]. The plant materials (0.5g) were homogenizedin 5mL of 0.1% (w/v) trichloroacetic acid (TCA) andcentrifuged at 10,000g for 20min. To 1mL aliquot of thesupernatant, 4mL of 0.5% thiobarbituric acid (TBA) in20% TCA was added. The mixture was heated at 95
◦
C for30minandquicklycooledinanicebath.Aftercentrifugationat 10,000g for 15min, the absorbance of the supernatantwas recorded at 532 and 600nm. The value for nonspecificabsorption at 600nm was subtracted. The concentrationof MDA was calculated using extinction coe
ffi
cient of 155mM
−
1
cm
−
1
.
2.6. Total Carbohydrates Content.
For determination of car-bohydratescontent,50mgofdry powderwasextractedusing10mL of ethanol:distilled water (8:2; v/v), and supernatantwascollectedaftertwicecentrifugationat1480g.Theresiduefrom ethanol extraction was subsequently used for polysac-charide extraction by boiling water[21]. Total carbohydrates
content was estimated by the method of Dubois et al. [22].
2.7. Hydrogen Peroxide Content.
.
Theplant materials (0.5g) were homogenized in 5mL of 0.1%(w/v) trichloroacetic acid (TCA) on ice and centrifuged at12,000g for 15min. To 0.5mL aliquot of the supernatant,1mL potassium phosphate bu
ff
er and 1mL KI was added.The absorbance of the supernatant was recorded at 390nm.
2.8. Statistical Analysis.
All analyses were performed basedon a completely randomized design. The data determinedin triplicate were analyzed by analysis of variance (
ANOVA
)using
SPSS
(version
9.05
). Each data point was the meanof three replicates (
n
=
3). The significance of di
ff
erenceswas determined according to Duncan’s multiple range test(DMRT).
P
values
<
0.05 are considered to be significant.
The Scientific World Journal 3
00.050.10.150.20.250.3Healthy Infected
F r e s h w e i g h t ( g P l a n t
−
1
)
aacb
(a)
00.010.020.030.040.050.060.070.080.09
D r y w e i g h t ( g P l a n t
−
1
)
Healthy Infectedaacb
(b)
606162636465666768697071Healthy Infected
R e l a t e d w a t e r c o n t e n t ( % )
EMFCtrlaaaa
(c)
Figure
1: E
ff
ect of EMF on the fresh weight (a), dry weight (b), and relative water content (c) in healthy and infected plants of
Citrusaurantifolia.
Vertical bars indicate mean
±
SE of three replicates. Di
ff
erent letters indicate significant di
ff
erences (
P <
0
.
05).
3.ResultandDiscussion
In order to determine the e
ff
ect of electromagnetic fieldon leaf growth and biochemistry in
Citrus aurantifolia
, wetreated the healthy and infected lime plants with electromag-netic field. The treatment of healthy and infected plants withelectromagnetic field a
ff
ected significantly the growth of thelime plants. Fresh and dry weight of leave in both healthy and infected plants increased under electromagnetic field(Figure1). The present data agree with the previous resultsreported on
Prunus maritime
,
Cucumis sativus
,
Raphanussativus
, and
Helianthus annuus
[24–26]. Relative water
content (RWC) decreased in healthy plants and increased ininfected plants under EMF stress (Figure1(c)).Protein content in leaves of both healthy and infectedplants increased significantly under EMF (Figure2). EMFdecreased slightly the content of total carbohydrates in bothhealthy and infected plants (Figure3). The reduction in
carbohydrates content is more prominent in healthy plantsthan that of infected ones.Free proline content increased significantly under EMFexposure (Figure4). However, enhanced levels of proline
accumulation may not be enough to maintain water balanceof the healthy plants under EMF treatment (Figure1(a)).Many plants accumulated proline as nontoxic and protectiveosmolyte under stress conditions [27–29]. Higher accumu-
lation of proline in lime plants under EMF may a
ff
ord itmuch protection against electromagnetic field. Although thepreciseroleofprolineaccumulationisstilldebated,prolineisalso considered to be involved in the preservation of cellularstructures, enzymes, and to exploit as a free radical scavenger[30,31].
Changes in lipid peroxidation serve as an indicator of theextent of oxidative damage under stress, with an unchanged
Weak EMFs affect Lime Trees - 2012 study
Abdollahi F, Niknam V, Ghanati F, Masroor F, Noorbakhsh SN. (2012). Biological Effects of Weak Electromagnetic Field on Healthy and Infected Lime (Citrus aurantifolia) Trees with Phytoplasma. Scientific World Journal. ;2012:716929. Epub 2012 May 2. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3354635/pdf/TSWJ2012-716929.pdf
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