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Molecular Pathogens
MP2011, Vol.2, No.2
http://mp.sophiapublisher.com
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of the foliage. The margins of yellow spots blend
gradually from light yellow into dark green in healthy
tissues. Mosaic spots on fruits, on the other hand,
were very similar to those on leaves but less
conspicuous. Symptoms of mosaic may not appear
uniformly over the entire plant. Similar mosaic,
malformation, and premature fruit dropping symptoms
were reported in infected fig plants (Martelli et al.,
1993; Serrano et al., 2004; Castellano et al., 2007).
The electron microscopy of ultra thin sections of
FMV-infected leaves judged by I-ELISA (previous
study) revealed the presence of so-called double
membrane bodies (DMBs) in parenchyma cells with
two types: Rounded to ovoid, 160~200 nm in size and
elongated; straight to slightly flexuous, up to or
exceeding 1 µm in length. Also accumulation of starch
grain was recorded as ultrastructure change of leaf of
F. carica
L. affected by FMV-infection.
These results were similar to that obtained by Serrano
et al. (2004) and Castellano et al. (2007). Comparable
large quasi-spherical DMBs of 100~150 nm in
diameter were located in the cytoplasm of parenchyma
cells. Electron dense median core particles were
observed as well. Serological methods depending on
the properties of surface viral protein have been used
to detect and identify plant viruses for many years
(Torrance and Jones, 1981). These methods were also
applied as a tool for the determination of the degree
relationships between the virus strains, and for the
taxonomy of plant viruses as well (Van Regenmortel
et al., 2000).
The sequence information for the
CP
genes is very
important criterion for the taxonomy of
potyviruse
s
(Shukla and Ward, 1989b). Consequently, universal
primers of
Potyviridae
(Chen et al., 2001) were used
and successfully amplified to 969 bp of
NIb
gene of
FMV in the infected tissue. Putative
potyviruses
were
reported from Croatia (Grbelja, 1983) and the
pathogen was assumed to be a member of the family
Potyviridae
by Brunt et al. (1996).
In addition, a novel primer set was developed to
amplify 374 bp within the
CP
gene from both infected
tissue and purified virus. This amplified product
represents more than 8 % of the whole genome. It is a
sufficient sequence to determine the species of the
virus and thus potentially to identify unrecognized
potyviruses
. One major problem with degenerate
primers is that the concentration of some permutations
in the mixture is so small due to their great
multiplicity; that amplification is effectively inhibited.
For any given viral RNA target, only a proportion of
the primer may participate in the initiation of high
efficiency extension in the early rounds of PCR. It was
believed that the redundancy of the CP1 and CP2 was
insufficient to cause this problem (Knoth et al., 1988).
The full length of
CP
gene from
Potyviridae
was
determined as 800 bp to 900 bp (Chen et al., 2001;
Fuji et al., 2003). The full length of
CP
gene from
FMV was about 864 bp. We can therefore conclude
that CP-like gene represents about 43% of the full
length of
CP
gene. Comparison of nucleotide
sequence of
CP
gene to all available sequences in the
GenBank created a significant homology with 14
CP
genes. The phylogenetic analysis indicated that our
FMV isolate was closely related to other FMV isolates,
especially the Arkansas and the Italian isolates.
Confirmatory results obtained using phylogenetic
analysis of
NIb
gene indicated that the FMV isolate
was closely related to the
NIB
genes of both Arkansas
and Italy FMV isolates. Subsequently, in order to take
a step toward classifying this unclassified virus, a
strategy used for genome sense investigation of FMV
was developed. The genome sense investigation
proved that the CP-like protein was expressed at the
molecular weight of 13.7 KDa on SDS-PAGE. This
step is a very good confirmation that FMV is a
positive sense RNA virus (
Potyvirus
). Similarly,
Jagadish et al. (1991) reported successful expression
of the full length of potyvirus coat protein in both
E.coli
and yeast. They also reported its assembly in
virus-like particles. Recently, some of the members of
Closteroviridae have been reported from the infected
fig trees in Italy. They are called fig leaf mottle-
associated viruses (FLMaV and FLMaV-2) (Elbeaino
et al., 2006, 2007). The latter was tentatively identified
as a putative species of genus Ampelovirus. The long,
flexuous, rod-shaped, virus-like particles in Turkish
fig leaves should be investigated as to whether or not