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, India , and Iran .
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
, proline, protein, and carbohydrates in phytoplasmas-infected lime plant under electromagnetic ﬁelds
2.1. Plant Materials and Electromagnetic Treatment.
L. Swingle cv. Keyline) were in-fected with the Witches’ broom disease of lime (WBDL)
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
, 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
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
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
4 (KV/m) and 3400
43 (mA/m),respectively . All measurements were conducted either infresh harvested tissue ground immediately after excision orfrom leaves quickly deep frozen in liquid nitrogen and keptat
C until the assay.
2.2. Plant Water Relations.
Leaf water content (WC) was cal-culated based on WC(%)
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
er (pH 7.0) containing 10mMEDTA, 2mM MgSO
, 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
C. Totalprotein content was measured by the spectrophotometricmethod of Bradford using bovine serum albumin (BSA)
as the standard.
2.4. Proline Content.
Free proline content was determinedaccording to Bates et al.  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 . 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
cient of 155mM
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. Total carbohydrates
content was estimated by the method of Dubois et al. .
2.7. Hydrogen Peroxide Content.
The content of hydrogenperoxide was determined according to Sergiev et al. 
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
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 (
). Each data point was the meanof three replicates (
3). The signiﬁcance of di
erenceswas determined according to Duncan’s multiple range test(DMRT).
0.05 are considered to be signiﬁcant.