Detection and identification of a new isolate of Grapevine fanleaf Virus naturally infecting Grapevine plants in Egypt using qReal Time-PCR

Grapevine fanleaf virus (GFLV) is a member of the genus Nepovirus in the family Comoviridae, a widely distributed virus responsible for grapevine ( Vitis vinifera ) degeneration. This virus causes serious economic losses by reducing grape crop yield. The Quantitative Real-Time Reverse Transcription-Polymerase Chain Reaction (qReal Time-PCR) assay was carried out on (GFLV) recovered from infected grapevines leaves at Alexandria, Egypt. A 606 bp fragment of the GFLV RNA-2 coat protein (CP) gene was amplified and then sequenced. Results of reactions of diagnostic hosts were observed on Gomphrena globosa , which developed systemic mottling, leaves twisting and necrotic spots during spring, whereas Chenopodium amaranticolor induced systemic mottling and leaf deformation, and its sap seemed relatively insensitive to the inhibitors of infection. Mottling of Glycine max was detected after inoculation, but inoculation of Nicotiana glutinosa didn't induce any symptoms. This study aimed to detect and identify a new isolate of GFLV-DA3 from Egypt using biological and molecular tools.


Introduction
Grape is one of the most popular fruits all over the world (Abido et al., 2013). In Egypt, grapes rank the second position in exportation after citrus. The total planted area of the vineyards in Egypt reached 167296 feddan with a production of 1370241 tons according to the latest statistics of the Ministry of Agriculture at (2009). The cultivars in Egypt cover approximately the whole season, these cultivars help in increasing exports to European, Arab and Asian countries (Ahmed et al., 2012). Grapevine fanleaf virus (GFLV) is a member of the genus Nepovirus in the family Comoviridae, and is a widely distributed virus responsible for grapevine degeneration. It causes serious economic losses by reducing yield, lowering fruit quality and substantially reducing the longevity of grapevines. Infected grapevines show a range of foliar Novel Research in Microbiology Journal, 2019 symptoms consisting of leaf deformation, yellow mosaic, vein banding, ring and line patterns and flecks (Martelli and Savino, 1990;Andret-Link et al., 2004). GFLV is specifically transmitted by the nematode Xiphinema index that feeds on growing root tips (Hewitt et al., 1958;Wyss, 2000). Virus strains were recovered by mechanical inoculation and maintained in Chenopodium quinoa (Raski et al., 1983;Bovey et al., 1990;Martelli and Savino, 1990). The genome of GFLV is bipartite and composed of two singlestranded positive-sense RNAs (Pinck et al., 1988). RNA1 encodes the poly protein P1 which matures into the VPg (viral protein genome-linked), the RNA polymerase, the proteinase and the NTP-binding protein. RNA2 encodes the polyprotein P2 that is subsequently cleaved into the movement protein and the 56 kDa coat protein (CP) reported by (Serghini et al., 1990;Gaire et al., 1999;Elbeaino et al., 2011).
The aims of the present study were; a)-to study the symptomology of some host ranges such as; Chenopodium amaranticolor, Gomphrena globosa, Nicotiana glutinosa and Glycin max, which were the most readily infected and common test plants routinely employed, b)-to partially characterize the GFLV-Egyptian isolate based on CP gene using; qRT-PCR, amplification of GFLV-CP gene using RT-PCR, sequencing and phylogenetic tree.

The natural plant source of GFLV
Naturally infected grapevines leaves were collected from several grapevine fields at New Borg El-Arab city, Alexandria, Egypt. The collected leaves were showing typical systemic symptoms of GFLV. Severe deformation of young grapevine leaves and conspicuous vein-clearing of these expanded leaves were observed.

Isolation of total RNA from leaves naturally infected with GFLV
Total RNA was extracted from grapevine leaves using RNeasy Mini Kit (QIAGene, Germany) according to the manufacturer's instructions, and then dissolved in diethyl pyro-carbonate treated water. The obtained RNA was dissolved in diethyl dicarbonatetreated water, incubated with DNase for 1 h at 37°C to remove any DNA residues, and then quantified using a Nano Drop 1000 spectrophotometer (Thermo Scientific, USA).

Amplification of the GFLV-CP gene using RT-PCR
For PCR amplification, a sense of the GFLV-CP gene (5`-GTGAGAGGATTAGCTGGT-3`) and the anti-sense (5`-AGCACTCCTAAGGGCCGT-3`) were designed from the CP gene located in the RNA2 of the GFLV infected leaves, according to Fattouch et al., (2001). The PCR amplification was carried out using 10 ng cDNA (1 µl), 10x buffer mix (12.5 µl), 10 poml /µl of each primers (2 µl), 5U Taq polymerase (0.25 µl, Bioline, Germany), and a final volume up to 25 µl with sterile water. The PCR reaction conditions were: initial denaturation at 95°C for 3 min. followed by 30 cycles; denaturation at 95°C for 30 sec, annealing at 50°C for 30 sec, and elongation at 72°C for 1 min. Final elongation at 72°C was done for 5 min. The PCR amplification products were separated by 2 % agarose gel electrophoresis according to Aseel et al., (2019

Sequencing, phylogenetic analysis of the GFLV-CP gene, and deduced amino acid sequence analysis
The amplified CP gene of the GFLV was sequenced using an automated sequencer (Macrogene Company, Korea), with forward universal primer. The nucleotide sequence was aligned using NCBI-BLAST, and then compared to the other Nepoviruses available in the GenBank database (http: //www. ncbi.nlm.nih.gov). For using amino acids sequence analysis, the DNA sequences were translated to deduced amino acids and aligned using the ClustalW2 Multiple Sequence program (Rice et al., 2000;Larkin et al., 2007). The alignment and phylogenetic analysis were carried out using the Molecular Evolutionary Genetics Analysis as described by Kawanna and Aseel, (2019).

Mechanical transmission of the GFLV in different herbaceous plants
Grape-leaf sap was manually inoculated to herbaceous plants by the method previously described by Cadman et al., (1960). Approximately, 0.2-0.5 g of young grape leaf was macerated in 5 ml (0.1 M) phosphate buffer (pH 7) and carborandum 600 mesh (Hamza et al., 2018). The herbaceous plants used include C. amaranticolor and Gomphrena globosa, which were the most readily infected plants and the common test plants routinely employed. All the herbaceous plants were kept in darkness for 1-2 d before inoculation, to increase their susceptibility to infection. After inoculation, the leaves were rinsed with tap water and then kept in daylight in an insect free glasshouse. The grapevine viruses were maintained in C. amaranticolor or Gomphrena globosa as sources of inoculum, their sap were prepared immediately before use. C. amaranticolor was also used for infectivity assays of the host plant. The collected virus sample was therefore inoculated into N. glutinosa, Glycin max, C. amaranticolor and Gomphrena globosa plants.

Quantitative expression of the GFLV-CP gene using qReal-Time-PCR
In the current study, measurement of the GFLV-CP gene expression was carried out using qReal-Time-PCR. Results recorded high expression level of about (~35-fold) of the GFLV-CP gene detected in the infected symptomatic leaves; however, no expression of this gene was observed with the healthy grapevine leaves as clear in Fig. (2).

Amplification of the GFLV-CP gene using RT-PCR, phylogenetic tree construction, and amino acid sequence analysis
The primers successfully amplified the cDNA product (606 bp) of the viral CP gene recovered from leaves infected with GFLV, whereas, no fragments are detected with the healthy plant (negative control) leaves, these results are shown in Fig. (3A). These results are in agreement with previous findings of Fattouch et al., (2001). Partial sequences of the GFLV-CP gene is aligned and compared with other Nepoviruses available in the GenBank database. The phylogenetic relationships were generated using the MEGA4 Bootstrab neighbor joining method. The Egyptian isolate of Grapevine fanleaf virus-DA3 is closely related with the Grapevine fanleaf virus GFLV-CP genes (AF4185790; AF304014, and JN585800) from Brazil; USA and Spain, with a nucleotide sequence identity of 94% (Fig. 3B).
The GFLV-CP sequences were aligned with different CP genes available in the GenBank database using the Clustal W2 Muliple Sequence Alignment program (1.83) software (Fig. 3C).   According to the previous results of Fazeli et al., (2000); Youssef et al., (2006), their putative GLRaV-1 sequence is closely related to the current Egyptian GLRaV-1 sequence with an identity of 95%. Moreover, analysis of the phylogenetic tree showed noticeable similarity with the newly Czech isolates sequenced from South Moravia, and relatively high dissimilarity from the rest of the analyzed isolates including the previously sequenced isolate HV5 from South Moravian region as reported by Eichmeier et al., (2010). In the current study, results of the deduced amino acid sequence showed variations of the GFLV isolate from Spain, i.e. H →E, A→ G, R→N, S→E, D→K and S→Q with substitutions in the Egyptian GFLV-DA3 isolate (Fig. 4A). The phylogenetic tree of the deduced amino acid sequence demonstrated that the GFLV-DA3 Egyptian isolate is closely related with Grapevine fanleaf virus GFLV from Spain with amino acid sequence identity of 87% (Fig. 4B). Similar results were obtained by Izadpanah et al., (2003), they observed that the inferred amino acid sequences were 96% similar. Where, many of the nucleotide differences either were silent or led to conservative amino acid substitutions.

Reactions of the diagnostic herbaceous hosts
The isolate of Grapevine fanleaf virus is transmitted from the infected plants, but the symptoms produced in C. amaranticolor and Gomphrena globosa plants are unlike those produced by isolates from other grapevines with yellow mosaic. Gomphrena globosa developed several symptoms including; systemic mottling, leaves twisting and necrotic spots during spring, whereas C. amaranticolor showed systemic mottling and leaf deformation. The virus seemed relatively insensitive to the inhibitors of infection present in the sap of C.
amaranticolor. On the other hand, Glycine max mottling appeared after inoculation (Fig. 5A-5D); however, N. glutinosa doesn't show symptoms after inoculation. The symptoms are developed on the diagnostic hosts on inoculation with the GFLV isolated from naturally infected vitis plants in accordance with Cadman et al., (1960); Raski et al., (1983);Bovey et al., (1990); Martelli and Savino, (1990). GFLV is sap transmitted to a limited range of hosts; these results are in agreement with the finding of Cadman et al., (1960);Dias, (1963). N. glutinosa reacted negatively with the GFLV isolate.

Conclusion
To our knowledge, this is the first study carried out in Egypt concerning the detection and identification of a new isolate of Grapevine fanleaf virus-DA3, from naturally infected grapevine field. The Real Time-qPCR approach demonstrated high specificity and sensitivity in the detection of this isolate of GFLV.