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Molecular Pathogens
MP2011, Vol.2, No.2
http://mp.sophiapublisher.com
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they belong to the family Closteroviridae.
Contradictory results were presented by Elbeaino et al.
(2009a), who proposed that FMV was classified as
genus Emaravirus, family Bunyaviridae (based on
BLAST analysis of sequence from the four RNA
segments). In parallel, Elbeaino et al. (2009b)
suggested that FMV is a negative-sense
single-stranded RNA virus belonging to the family
Bunyaviridae (based on BLAST analysis of sequence
from the two largest RNA segments).
3 Materials and methods
3.1 Sample collection
In summer 2008, 140 samples from naturally infected
fig (
Ficus carica
L.) plants exhibiting characteristic
fig mosaic virus-like symptoms (chlorotic spotting of
leaves) were collected from different fields in the
north coast of the western desert, which, extends from
the west of Alexandria to the Marsa Matrouh along
250 km. Healthy material was obtained from seedling,
or by shoot-tip tissue culture.
3.2 Electron microscopy
Pieces of healthy and infected fig mosaic leaves were
prepared for transmission electron microscopy (TEM)
using standard procedures (Martelli and Russo, 1984).
Briefly, samples for TEM were excised from infected
fig leaves, fixed immediately in a solution of 3%
glutaraldehyde in 50 mM phosphate buffer (pH 7.2),
and kept overnight at 4
℃
. Samples were washed in
the same buffer and post-fixed in 1% osmium
tetroxide (OsO4) in the same buffer for 2 hours at
room temperature. Following osmium tetroxide
fixation, samples were dehydrated in a series of
increasing acetone concentrations. Dehydrated
samples were subsequently embedded in an Epon
araldite mixture (Medina et al., 2003; Soylu et al.,
2005). Ultra-thin sections (70~90 nm) were cut with
an Ultracut E microtome (Reichert, Milton Keynes,
UK) using diamond knives (Diatome, Bienne,
Switzerland). Sections were then routinely mounted
for staining on formvar-coated, 200 mesh copper grids
(Aldrich, Dorset, UK). Grid-mounted sections with
silver-gold interference color were stained in drops of
4.5 % uranyl acetate. After treatments, grids were
washed in DH2O and further stained in drops of
Reynold’s lead citrate (Roland and Vian, 1991). The
ultrathin sections were examined with transmission
electron microscope, JEOL-CX100 operating at 80
KV (The electron microscope unit, Faculty of Science,
Alexandria University, Egypt).
3.3 Extraction of total RNA from plant tissues and
purification of viral particles
dsRNA isolation from fig tissue (healthy & infected )
were done using RNeasy Mini Kit according to
manufacturer's instructions (QIAGEN, Germany),
where, the viral RNA was isolated from the purified
viral particles using QIAamp viral RNA isolation kit
(QIAGEN, Germany) according to the manufacturer’s
instructions. The RNA was dissolved in DEPC-treated
water, quantitated spectrophotometrically and analyzed
on 1.2% agarose gel.
3.4 Reverse transcription-polymerase chain reaction
(RT-PCR)
Reverse transcription reactions were performed in
reaction 25 µl. The reaction mixture containing 2.5 µl
of 5x buffer with MgCl2, 2.5 µl of 2.5 mM dNTPs, 4
µl from oligo (dT) primer (20 pmol/ µl), 2 µg RNA
and 200 U reverse transcriptase enzyme (MLV,
Fermentas, USA). RT-PCR amplification was
performed in a thermal cycler (Eppendorf, Germany)
programmed at 42°C for 1 hour, at 72°C for 10
minutes, and the cDNA was then stored at -20°C until
used.
3.5 Detection of
fig mosaic virus
using universal
primer of Nuclear Inclusion Body (
NIB
) of
Potyvirus
Universal primers (S primer and M
4
) of
potyviridae
(Chen et al., 2001) were used to detect FMV in the
infected tissue. 2 µl of the amplified cDNA were
added to 2.5 µl Taq polymerase buffer 10x (Promega,
Madison, USA), 2.5 µl of 25 mM MgCl
2
, 2 µl of 2.5
mM dNTPs, 2 µl of 20 pmol/ul of each primer (S
primer GGXAAYAAYAGYGGXCAZCC and M4
GTTTTCCCAGTCACGAC) and 0.2 µl Taq polymerase
(5 U/ µl) in a final reaction volume of 25 µl. The
PCR conditions were; Initial denaturation at 95°C for
5 minutes, followed by 34 cycles at 95°C for 1 minute,
at 47°C for 1 minute, and at 72°C for 1 minute. Final
extension at 72°C was done for 10 minutes.
Amplification products were electrophoresed in 1.5%