Incidence of Fig Leaf Mottle-associated Virus and Fig Mosaic Virus in Eastern
Province of Saudi Arabia
Fig plant, Ficus carica L., is grown in Saudi Arabia and is being affected
by fig plant mosaic diseases (Fig leaf mottle-associated virus, Fig mosaic virus).
The main symptoms are chlorotic mottling, blotching and various types of leaf
deformation. Samples were collected, with consideration of the economically
importance and distribution of the cultivars, from different areas of Hofuf
Saudi Arabia. Each sample was consisted of 10-15 leaves. Samples were labeled
and stored in plastic bags at 4°C; then transferred to the laboratory, for
total nucleic acids (TNAs) extraction. One hundred milligram of leaf veins and
or cortical scrapings were used for extraction. Samples were macerated in 1
mL of grinding buffer. TNAs were recovered with a silica-capture procedure and
stored at -20°C till used. The 8-10 μL of TNA extracts were mixed with
1 μL random hexamer primer, (Boehringer Mannheim, GbmH) (0.5 μg μL-1).
RT-PCR assay of leaves extracts of infected fig accession using specific primers
gave positive results and non with FLMaV-2. Mixed infection of FLMaV-1 and FMV
were found. To our knowledge this is the first record and identification of
FLMaV-1 and FMV in Saudi Arabia. Further studies are needed to investigate the
fig mosaic disease throughout the country.
June 20, 2011; Accepted: July 27, 2011;
Published: October 25, 2011
Fig plant, Ficus carica L., Brown Turkey, is grown mainly as individual
trees in farm, rarely in specialized orchards in Hofuf, Saudi Arabia. A wide
range of discolourations, i.e, various patterns of chlorotic mottling and blotching,
vein banding, vein clearing and malformations of the leaves are commonly observed
on fig tree throughout Saudi Arabia. Fig mosaic virus is a graft-transmitted
disease. It is also transmitted by the eriophyid mite Aceria ficus (Condit
and Horne, 1933; Flock and Wallace, 1955). Recent
studies have reported that tissues of infected fig leaves have shown particles
of this virus those are likely to be intracellular enveloped structures (Bradfute
et al., 1970; Castellano et al., 2007;
Elbeaino et al., 2007a, b).
Other viruses associated with fig mosaic diseases were found such as fig leaf
mottle associated virus 1 (FLMaV-1) and fig leaf mottle associated virus 2 (FLMaV-2)
that are members of Closteroviridae (Elbeaino et al.,
2006; Elbeaino et al., 2007a, b).
This study is aimed at identification of such mosaic-causing viruses i.e. FLMaV-1,
FLMaV-2 and FMV in Saudi Arabia.
MATERIALS AND METHODS
Collection of samples: The main symptoms as described by Castellano
et al. (2007) were chlorotic mottling, blotching and various types
of leaf deformation. A survey was carried out in autumn seasons of 2008-2009.
||Map of the area where survey has been conducted in Hofuf,
Samples were collected, with consideration of the economic importance and
distribution of the cultivars, from different areas of Hofuf Saudi Arabia (Fig.
1). Each sample was consisted of 10-15/leaves. Samples were labeled and
stored in plastic bags at 4°C then transferred to the laboratory.
Extraction of total nucleic acids (TNAs): TNAs were extracted from leaf
veins and/or cortical scrapings of fig samples, 100 mg of tissue were macerated
in 1 mL grinding buffer (6 M guanidine isothiocyanate, 0.2 M sodium acetate,
1 M potassium acetate, 0.025 mM EDTA, 25% PVP-40). TNAs were recovered with
a silica-capture procedure (Foissac et al., 2001);
stored at -20°C, and then used as described by Elbeaino
et al. (2009 a,b).
Molecular detection of Fig mosaic disease in tissue extract: Fig samples
were assayed by RT-PCR using the following specific primers (Elbeaino
et al., 2006, 2009):
||LMaV- N17s: 5- CGTGGCTGATGCAAAGTTTA-3
||FLMaV-2 F3-s: 5'- GAACAGTGCCTATCAGTTTGA TTTG-3'
||F3-a: 5'-TCCCACCTCCTGCGAAGCTAG AGAA-3'
||FMV E5-s: 5-CGGTAGCAAATGGAATGAAA-3 E5-a
From 8-10 μL of TNA extract were mixed with 1 μL random hexamer primer,
(Boehringer Mannheim, GbmH) (0.5 μg μL-1), denatured at
95°C for 5 min and quickly chilled in ice. Reverse transcription reaction
was done for 1 h at 39°C by adding 4 μL M-MLV buffer 5x, 2 μL
of 10 mM DTT, 0.5 μL f 10 mM dNTPs, and 200 units Moloney Murine Leukaemia
virus (M-MLV) reverse transcriptase (Bethesda Research Laboratories USA) in
a final volume of 20 μL. For RT-PCR with the specific primers detection,
2.5 μL cDNA mixture were submitted to PCR amplification by adding 2.5 μL
of 10xTaq polymerase buffer (Promega Corporation, USA), 1.5 mM as final concentration
of MgCl2, 0.5 μL of 10 mM dNTPs, 0.5 μL of 10 μM forward
and reveres of each specific primers, and 0.2 μL of Taq polymerase (5 unit
μL-1) in final volume of 25 μL. PCR products were electrophoresed
in agarose gels, and visualized by UV transillumination.
RESULTS AND DISCUSSION
Collection of samples: As a survey conducted from different areas of
Hofuf, Saudi Arabia; fig orchards and nursery were infected about 4%. Most of
nursery fig trees were brought from Lebanon, Jordan and Syria. Symptoms were
similar to those described by Castellano et al. (2007),
chlorotic mottling, blotching, vein banding, vein clearing and malformations
(Fig. 2, A-C). Symptoms were shown very clear in fig trees;
however, sometimes in summer season same symptoms were not shown. This indicates
that high temperature plays role in the development of these viruses.
Molecular detection of Fig mosaic disease in tissue extract: Fig mosaic
disease virus was amplified from leaf samples showing typical symptoms (Castellano
et al., 2007). Only infected fig trees were PCR-positive where no
PCR products were obtained from healthy fig tree (Martelli
et al., 1993; Elbeaino et al., 2007a,
b, 2009a, b).
RT- PCR assay of leaf extracts of infected fig accession using FLMaV-1 and FMV
specific primers had gave positive results and non with FLMaV-2. The three sets
of primers amplified DNA fragments of 350 and 302 bp for FLMaV-1 and FMV, respectively
(Fig. 3a, b). However, mixed infections
were obtained of FLMaV-1 and FMV from Fig mosaic disease (Elbeaino
et al., 2007a).
||(a) Mottle and mosaic symptoms on naturally-infected leaf.
(b) Ring spots in fig fruit of naturally infected plant and (c) Mosaic viral
symptoms on fig leaf during the heat of summer in Hofuf
||Gel electrophoresis of RT-PCR assay showing a product of 350
and 302 bp for FLMaV-1 and FMV, respectively. (a) lane, 2 infected samples
by FLMaV-1; lanes 1 and 3 healthy samples; lane, 4 positive control. (b)
lanes 1, 2, 3 infected samples by FMV; lane 4 negative sample; lane, 5 positive
control. M, 1 kb marker
FLMaV-1 and FMV have been recorded in Italy (Elbeaino et
al., 2006, 2009a, b),
Albania, Algeria, Syria, Chile, Greece, Mexico and South Africa (Castellano
et al., 2007), Lebanese (Elbeaino et al.,
2007b) and Tunisa (Nahdi et al., 2006). To
our knowledge this is the first record of identification of FLMaV-1 and FMV
in Saudi Arabia. Further studies are needed to investigate the fig mosaic disease
throughout the country.
This research was supported by Deanship of Scientific Research, King Faisal University, Saudi Arabia. The author thanks Dr. Elbeaino, Istituto Agronomico Mediterraneo, Bari, Italy for his assistance and support.
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