The first known reports of fig mosaic disease (FMD) were submitted by Condit in 1922 and Swingle in 1928 (cited in Alfieri, 1967), but the first critical study was conducted by Condit and Horne (1933). The disease has been widespread in several fig-growing countries, including Egypt. Although it was not sap- or seed-transmissible (Martelli et al., 1993; Elbeaino et al., 2006), successful transmission of the disease by an eriophyid mite, Aceria ficus Cotte, has been reported by Flock and Wallace (1955). The aetiology and mechanical transmission of the disease is still uncertain (Martelli et al., 1993). The disease was thought to be of viral origin since ultrastructural observations revealed the occurrence of intracytoplasmic enveloped spherical bodies in infected fig cells (Bradfute et al., 1970; Plavsic and Milicic, 1980; Appiano et al., 1990). In the following years, the agents of the disease were called “disease-associated bodies” (DABs) or round to ovoid double membrane bodies (DMB), which had an envelope consisting of a unit membrane approximately 12 nm thick and 90~200 nm in diameter in the cytoplasm of parenchyma cells differed in shape and size (Bradfute et al., 1970; Martelli et al., 1993; Appiano et al., 1995). DMBs contain proteinaceous material, fine fibrils, and are often next to aggregates of convoluted electron-dense filamentous elements that contain carbohydrates and are partially digested by pronase (Appiano and Conti, 1993; Castellano et al., 2007). They are also insensitive to tetracycline (Martelli et al., 1993). Because of this and of their cytochemical and ultrastructural properties, DMBs were suggested to represent possible particles of an uncharacterized virus (Martelli et al., 1993; Ahn et al., 1996).
Putative potyviruses were reported from Croatia (Grbelja, 1983), and then the pathogen was assumed to be a member of the family Potyviridae by Brunt et al. (1996). Some double membrane-bound bodies (DMBs) and rod-shaped virus particles (720 nm in size) showing a tail of 230, which might be responsible for the disease, were described as possible agents of the disease (Serrano et al., 2004). Double stranded RNAs (dsRNA) with a size ranging from 0.6 to 6.6 kb were obtained from infected trees in Turkey (Açýkgöz and Timur Döken, 2003). The main objective of the present work is to identify and characterize an Egyptian isolate of the fig mosaic virus (FMV).
1.1 Disease Incidence
Fig mosaic symptoms were observed in all the fields surveyed. The symptomatology identification of FMV was recorded as: light chlorotic spotting, mottling, extensive chlorosis along the veins and leaf malformation as shown in Figure 1.
Figure 1 The observation of Fig mosaic symptoms
1.2 Electron microscopy and Cytopathological effects
Electron micrographs of ultrathin sections prepared from healthy F. carica L. leaves, represented in Figures 2, exhibited normal cell structures, while those prepared from FMV-infected F. carica L. leaves revealed many cytopathological effects. Figures 3 show starch grains accumulation inside the chloroplasts. Two types of intracytoplasmatic electron-dense bodies with a double membrane (DMBs) were observed in parenchyma and subepidermical cells of fig mosaic leaves, always presented in rounded to ovoid 160~200 nm in size and elongated, straight to slightly flexuous up to or exceeding 1µm in length. Long elongated and flexuous virus-like particles surrounding the chloroplast in parenchyma cell are shown Figure 4.
Figure 2 The results of electron micrographs of ultrathin sections from F. carica L.
Figure 3 Starch grain accumulation by electron micrographs of ultrathin sections F. carica L.
Figure 4 Electron micrographs of ultrathin section sections from FMV-infected leaf of F. carica L. showing profile of long elongated and flexuous virus-like particles
1.3 Detection of fig mosaic virus using the NIB gene universal primer of Potyviruses
Universal primers of the Nuclear Inclusion Body of the potyviridae were designed by (Chen et al., 2001) were used and successfully amplified 969 bp of NIb gene of FMV in the infected tissue. This band was successfully amplified from RNA extracted from purified virus as well (Figure 5). Sequence and sequence analysis for the amplified 969 bp revealed that this DNA fragment was amplified within the nuclear inclusion body (NIb). This sequence was deposited in GenBank under accession number (Acc# GQ871933). Phylogenetic analyses, shown in Figure 6A, revealed that the NIb, a phylogenetic tree, was generated from sequence data of 14 virus isolates by UMPGADA distance analysis with maximum sequence difference of 0.8. The maximum nucleotide sequence divergence was exhibited in lineage I. Meanwhile, the FMV isolates appeared in the other lineage as monophyletic sister clade, as shown in Figure 6B. The phylogenetic analysis indicated that the Egyptian FMV isolate was closely related to Arkansas (Acc# FJ769161) and Italy (Acc# FM864225) fig mosaic viruses with similarity 56%. When the phylogeny was constructed based on the deduced amino acids sequences, the similarity between the Italian fig mosaic virus and ours (Egyptian fig mosaic virus) was increased up to 55%. Meanwhile, the Arkansas isolate was grouped with other Italian isolates (FM991954 and FM992851) with similarity 42%.
Figure 5 PCR products of the fig mosaic virus coat protein gene and Fig Mosaic Virus NIB gene
Figure 6 Phylogenetic analysis based on the nucleotide and the deduced amino acid sequences
1.4 Nucleotide sequence and sequence analysis of CP gene
RT-PCR using degenerate primer set designed (based on amino acid conserved region of 67 mosaic virus coat protein sequence) with an amplification product of 374 bp was obtained (Figure 5). The sequence was deposited in GenBank under accession number (Acc# GQ288368). This band was successfully amplified from RNA extracted from both symptomatic tissues and purified viruses (Figure 5). The sequence of this fragment showed one ORF within the FMV coat protein (CP) gene. Sequence alignment showed that the site of this fragment was about 1~146 codons after the starting codon, AUG of the FMV coat protein gene.
An ORF that could encode a polypeptide of 124 amino acids was detected. This deduced polypeptide contains 15 strongly basic, 14 strongly acidic, 35 hydrophobic, and 33 polar amino acids. The calculated molecular mass of the putative polypeptide is 13.67 kDa. Phylogenetic tree construction was based on the deduced amino acid sequences for the obtained CP with other coat protein genes for 14 Fig mosaic viruses, as depicted in Figure 7. The neighbor-joining distance analysis with maximum sequence difference of 1.2 and the topology yielded four distinct lineages that were similar to the Arkansas mosaic virus (Acc# FJ769161, with identity 65%) and to the Italian mosaic virus (Acc# FM864225, with identity 65%).
Figure 7 Phylogenetic analysis based on the deduced amino acid sequences showing the genetic relationship between the NIb genes of FMV with those of selected other viruses
1.5 Recombinant coat protein and SDS analysis
The recombinant cells were harvested and hydrolyzed; and the recombinant protein was separated on 12% SDS-PAGE. The protein with molecular weight of 13.7 kD was presented only in the recombinant bacterial cells compared with the non recombinant ones (Figure 8).
Figure 8 Agarose gel electrophoresis and SDS-PAGE of the cloning coat protein gene and the Fig Mosaic Virus coat protein gene
According to FAO (2008), the Mediterranean basin area is known to produce 80% of global production of fig. Egypt provides 27%, Turkey 11% and Europe 15%, in addition to the other countries. Egypt, Turkey, Iran, Algeria and Morocco were considered the top five fig-producing countries in the world. Fig mosaic disease (FMD) is an economically important disease that occurs naturally, wherever the common edible fig (Ficus carica L.) grows. It was observed in 14 different geographical origins of the world: Spain, England, Albania, Cyprus, Greece, Turkey, Israel, Yemen, Egypt, Tunisia, Algeria, Morocco, Italy and California (Martelli et al., 1993; Ahn et al., 1996). Among important viruses that caused devastating losses by reducing either the yield and/or quality of fig fruits is fig mosaic virus (FMV). For this purpose, the present investigation aimed to identify unidentified isolate of this virus based on different biological, serological and molecular tools, in order to provide a powerful diagnostic tool for early detection of FMV in infected tissues. In the present study, symptomatology identification of the FMV causes symptoms on both leaves and fruits. The mosaic spots were distinctly yellow on leaves, contrasting with normal green color 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) andCastellano 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 (Ahemaidan, 2000). 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 potyviruses (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 KD 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 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).
In this paper, the biological, ultrastructural, and molecular characterization of Egyptian isolate of FMV was described. Furthermore, cloning and sequencing of a conserved region in CP gene were performed. Further studies to classify the isolate as putative potyvirus were done. The availability of CP products would be helpful in studies concerning ELISA, PCR, and other related molecular techniques. Additionally, a novel primer set was developed to amplify 374 bp within the CP gene from both infected tissue and partial purified virus of an Egyptian FMV isolate. We suggest that it may be useful to monitor the distribution of FMV, and the release of wild type as well as the genetically engineered FMV.
4 Materials and Methods
4.1 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.
4.2 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 4â„ƒ. 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).
4.3 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.
4.4 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 42â„ƒ for 1 hour, at 72â„ƒ for 10 minutes, and the cDNA was then stored at -20â„ƒ until used.
4.5 Detection of fig mosaic virus using universal primer of Nuclear Inclusion Body (NIB) of Potyvirus
Universal primers (S primer and M4) 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 MgCl2, 2 µL of 2.5 mM dNTPs, 2 µL of 20 pmol/µL of each primer (S primer GGXAAYAAYAGYGGXCAZCC and M4 GTTTTCCCA GTCACGAC) and 0.2 µL Taq polymerase (5 U/ µL) in a final reaction volume of 25 µL. The PCR conditions were; initial denaturation at 95â„ƒ for 5 minutes, followed by 34 cycles at 95â„ƒ for 1 minute, at 47â„ƒ for 1 minute, and at 72â„ƒ for 1 minute. Final extension at 72â„ƒ was done for 10 minutes. Amplification products were electrophoresed in 1.5% agarose gels running in 0.5x TBE buffer.
4.6 Amplification of a conserved region within the coat protein gene of FMV
Based on the amino acid conserved region of 67 virus coat protein sequences of potyviruses available in Genbank, two degenerate primers were designed and used to amplify cDNA fragment within the coat protein gene of the infected tissues. The forward and reverse primers were designated as CP1 and CP2, respectively. CP1 was (5'-ZAY GGX GAX GAZ CAZ GTG-3') and CP2 was (5'AAZ GCX GCZ GCX ATY AAY -3'). The PCR reaction conditions were initial denaturation at 95 °C for 5 min, followed by 34 cycles at 95â„ƒ for 1 min, 60â„ƒ for 1 min and 72â„ƒ for 1 min. Final extension at 72â„ƒ was done for 10 min. Amplification products were visualized in 1.5% agarose gel run in 0.5x TBE buffer.
4.7 PCR purification, sequencing and sequencing analyses
PCR products were purified using PCR clean up column kit (Maxim biotech INC, USA) according to manufacturer's instructions and then sequenced using forward primer. Sequencing was performed using BigDye® Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA) and model 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Analysis of nucleotide sequences was carried out using Blast search. But for sequence alignment, the obtained sequence was aligned with the published ones using ClustalW (1.83) according to Thompson et al., 1994. Phylogenetic analysis was performed using MEGA4 program (Tamura et al., 2007).
4.8 Cloning, subcloning and gene expression of the amplified CP
To examine if the extracted viral RNA is a negative strand or a positive one, the purified PCR product corresponding to the CP gene fragment was cloned in PCR-TOPO vector with TOPO TA cloning kit (Invitrogen, USA). Subcloning was performed into pPROEX HTa expression vector using pPROEXTM HTa Prokaryotic Expression System kit (Life technologies, USA). Bacteria were grown at 37â„ƒ for 3 h, induced by the addition of 1 mM IPTG, incubated at 37â„ƒ for 5 h and then the cells were harvested, hydrolyzed and partial protein was obtained according to Mellor et al. (1983). Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out using the discontinuous buffer system as described by Sambrook et al. (1989) in order to demonstrate the expression level of the coat protein gene in the induced and non induced recombinant bacteria.
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