Rolling circle amplification-based detection and ... · Rahmany, 2015; Farrag et al., 2014; Lapidot et al., 2014). These techniques require a prior knowledge on nucleotide sequences

Middle East Journal of Applied Sciences ISSN 2077-4613

Volume : 09 | Issue :01 |Jan.-Mar.| 2019 Pages: 155-166

Corresponding Author: Mohamed Hassan, Agricultural Botany Department, Faculty of Agriculture, Fayoum University, Egypt. E-mail: [email protected]

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Rolling circle amplification-based detection and recombination analysis of Squash leaf curl virus in Egypt

Mohamed Hassan

Agricultural Botany Department, Faculty of Agriculture, Fayoum University, Egypt.

Received: 05 Jan. 2019 / Accepted 10 Mar. 2019 / Publication date: 25 Mar. 2019 ABSTRACT

Squash leaf curl virus (SLCV) is an emerging virus in Egypt over the last decade. Virus evolution

and recombination events in begomoviruses have played a role in the emergence of new strains or species. One promising generic detection approach is rolling circle amplification (RCA). RCA is exploited as a sequence-independent amplification strategy that amplifies circular DNA with high fidelity, including begomoviruses. We detected Egyptian isolates of SLCV by combining rolling circle amplification (RCA) with restriction fragment length polymorphism (RFLP). Also, we assessed the genetic diversity of two different isolates collected from open fields at Fayoum and Bani Suef governorates. Enzymatic digestion of RCA product with the restriction enzyme HpaII, revealed diverse band patterns in 22 squash samples. Representative viral full-length DNAs of SLCV isolates denoted (SLCV-Sq-EG1 and SLCV-Sq-EG2) were sequenced. Sequence analysis revealed footprints of recombination in their genomes underscoring the risk of generating new SLCV strains could adapt to new plant hosts in Egypt. Keywords: SLCV, RCA, RFLP, diversity, recombination, diagnostic, Egypt

Introduction

Geminiviridae is classified by the International Committee on Taxonomy of Viruses (ICTV) into

nine genera based on their genome arrangement, insect vector, and host range (Zerbini et al., 2017). Begomovirus is the largest genus in the Geminiviridae, with more than 150 species reported to date (Zerbini et al., 2017). Numerous begomoviruses, including Squash leaf curl virus (SLCV), have emerged as devastating pathogens, causing major economic losses of economically important cucurbits (Ali-Shtayeh et al., 2014; Lapidot et al., 2014).

In 2006, SLCV was first reported in Egypt and caused severe symptoms in squash fields (Idris et al., 2006). In parallel, SLCV has been emerging in Israel, Jordan, Lebanon, and Palestine (Lapidot et al., 2014). In Egypt, SLCV extended its cucurbits host range to include common bean, tomato, pepper and eggplant causing leaf deformation and yield reduction (EL-Rahmany, 2015; Farrag et al., 2014; Awad et al., 2018). SLCV transmitted by B. tabaci in a persistent circulative manner (Ghanim, 2014).

Other begomoviruses, like Watermelon chlorotic stunt virus (WmCSV), it was predicted that the virus will expand its range to Egypt due to frequent migration of cucurbits-infecting begomoviruses between Middle Eastern nations (Ali-Shtayeh et al., 2014; Al-Saleh et al., 2014; Khan et al., 2012; Lapidot et al., 2014). Co-infection of tomato breeding lines with SLCV, WmCSV and Tomato yellow leaf curl virus (TYLCV), was reported in Jordan (Ahmad et al., 2013).

Previous studies on detection and variability of SLCV from Egypt were PCR-based methods using either specific or degenerated primers (Abdel-Salam et al., 2006; El-Dougdoug et al., 2009; EL-Rahmany, 2015; Farrag et al., 2014; Lapidot et al., 2014). These techniques require a prior knowledge on nucleotide sequences of the viruses to be studied, moreover specific PCR tends to amplify only particular variants which could lead to bias in diversity study.

To ascertain presence of new begomoviruses or recombinants, sequence-independent detection methods like rolling circle amplification (RCA) are very useful (Ali-Shtayeh et al., 2014; Haible et al., 2006; Jeske, 2018; Kushawaha et al., 2018).

The aim of this study is to identify begomoviruses associated with squash leaf curl disease (SLCD) in Egypt using RCA/RFLP technique and to gain insight into the evolution of the SLCV isolates.

Middle East J. Appl. Sci., 9(1): 155-166, 2019 ISSN 2077-4613

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Materials and Methods Sampling

Virus-like symptoms were observed on squash plants (Cucurbita pepo cv. Eskandrani) grown in Fayoum and Bani Suef governorates in Egypt. Diseased plants exhibited symptoms including leaf mottling, curling, and stunting (Fig. 1). In addition, high population of whitefly (B. tabaci) was observed infested diseased squash plants. To identify the etiology of the disease, a total of twenty-two leaf samples (12 from Fayoum and 10 from Bani Suef governorates) were collected from symptomatic squash plants, and samples were processed directly in the laboratory.

Detection of begomoviruses by PCR

Total nucleic acids (TNAs) were extracted from 100 mg of symptomatic squash leaves using hot SDS method (Doyle, 1990). For detection using PCR, the degenerate primer pair AVcore/ACcore was used as described by (Wyatt and Brown, 1996). Nucleic acids obtained from healthy squash plants were used as negative control. PCR reactions were performed in PCR thermal cycler (Biometra, Germany). Each reaction contained 100 ng of sample DNA, 0.25 mM dNTPs, 0.25 mM MgCl2, 2.5 mM of each primer, 0.5U of Taq DNA Polymerase. The amplification program was as follows: an initial denaturing step at 94◦C for 3 min, followed by 35 cycles of 94◦C for 1 min, 55◦C for 40s and 72◦C for 1 min, and a final extension at 72◦C for 10 min. Amplified PCR products were resolved on 1 % agarose gel stained with ethidium bromide (0.5 mg/ml), and photographed.

Rolling circle amplification (RCA)

Aliquots of TNAs were subjected to Templi PhiTM Amplification Kit according to the manufacturer’s instructions (GE Healthcare, Germany). Briefly, 2ul (10-20 nanogram) of total DNA was dissolved in 5ul of sample buffer, denatured for 3 min at 95 C and cooled down on ice for 1 min. Then 5ul reaction buffer and 0.2ul enzyme mix were added to the mixture. Amplification was carried out for 16–20 h at 30◦C and the reaction was terminated by heating for 10 min at 65◦C to inactivate the enzyme (Laney et al., 2012).

Restriction fragment length polymorphism (RFLP)

The RFLP analysis was carried out by digesting 1ul of the RCA product with the restriction enzyme HpaII (New England Biolabs). The reaction was performed at 37◦C for 2 h, then the reaction was inactivated by treatment for 20 min at 65◦C, according to the supplier’s recommendation. DNA fragments were resolved on 2 % agarose gel following standard protocols and were visualized by ethidium bromide staining.

Cloning and sequencing of RCA fragments

RCA products of samples which show different banding pattern using RCA/RFLP (samples 5 and 8; Fig 2B) were partially digested with the 0.4U of enzyme Sau3AI for one minute (New England Bio-labs). Then the reaction was inactivated by fast heating for 10 min at 65◦C (Laney et al., 2012). Fragments at the range of 2.5-3 kbp were gel purified and inserted into BamHI-cut pGreen0029 plasmid as described by (Wyant et al., 2011). Then, recombinant plasmids were transformed into E. coli DH5α cells. Recombinant plasmids with the expected insert more than or equal to 2.5-3kbp were sequenced using a universal M13 primer in facilities of Macrogen Inc., (South Korea).

Sequence analysis

Complete sequences of DNA-A and DNA-B components were assembled using a Contig Assembly Program (CAP) implemented in BioEdit software (Hall, 1999). Sequence alignments were performed using ClustalW in BioEdit software. The open reading frames (ORFs) encoded by SLCV were identified with the ORF finder programs (http://www.ncbi.nlm.nih.gov/gorf/gorf.html).

Phylogenetic analyses were conducted on matrices of aligned sequences using the Neighbour-joining method and bootstrap options of MEGA7 (Kumar et al., 2016). Virus names and accession numbers are listed in Table 1.

http://www.ncbi.nlm.nih.gov/gorf/gorf.html


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Recombination analysis The potential recombinants events were tested in the genome of the representative SLCV isolates

infecting different hosts and geographical regions. DNA-A and DNA-B full genome sequences were aligned using ClustalW implemented in BioEdit software (Hall, 1999). Putative recombination breakpoints were assayed in aligned sequences on RDP v.4 software using GENECONV, BOOTSCAN, MAXCHI, SISCAN, 3SEQ, LARD, and RDP algorithms at default settings (Martin et al., 2015). Events supported by at least six or more algorithms with a probability value threshold of ≤0.05 were considered plausible recombination events Results Symptomatology

Samples collected from Fayoum and Bani Suef governorates showed symptoms similar to those described to be caused by SLCV (Abdel-Salam et al., 2006). Infected plants exhibited symptoms including leaf mottling, curling, and deformation (Fig. 1A-C). In autumn 2016, disease incidence reached 90% in squash fields surveyed (number of symptomatic plants/ 100 plant observed). Notwithstanding, all the infected plants were heavily infested with B. tabaci. Analysis of the collected squash samples by PCR using degenerate primers revealed that 20 out of 24 samples tested were positive for begomoviruses (data not shown).

Fig. 1: (A, B and C) Symptoms exhibited on squash plants naturally infected with SLCV showing a

severe leaf curling and mottling.

Diagnostic of SLCV Egyptian isolates using RCA-RFLP Twenty-four samples were subjected to RCA amplification using the bacteriophage Ø DNA

polymerase enzyme. 1μl of RCA product was digested with the restriction enzyme HpaII which cuts frequently all known begomoviral components as described previously (Haible et al., 2006; Homs et al., 2008; Schubert et al., 2007).

Twenty-one out of twenty-two squash samples collected from Fayoum and Bani Suef governorates found to contain a small circler DNA molecules using RCA/RFLP (Fig. 2). RFLP analysis of RCA products using HpaII restriction enzyme produced eight bands, however, samples number 5 and 8 revealed different polymorphic pattern (Fig. 2). In all cases, the sum of fragment sizes in each positive sample approaches 5 Kbp, which indicates the bipartite nature of detected begomoviruses (Fig. 2).

Sequence comparisons

Except for samples number 5 and 8 (Fig. 2), all positive samples show banding patterns similar to those reported for SLCV isolates from Palestine (Ali-Shtayeh et al., 2014). To ascertain the difference was observed in RCA/RFLP banding pattern in samples number 5 and 8, both samples were subjected to limited enzymatic digestion using Sau3AI restriction enzyme, which yielded partial dimers as a smear (Fig. 3). To ensure cloning the full genome of viral components, the fragments in the range of 2.5 to 3kbp were cloned and sequenced using M13 universal primers and a primer walking strategy. Full


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genome sequences of two different isolates were assembled. Sequenced SLCV isolates were denoted as SLCV-Sq-EG1 and SLCV-Sq-EG2, both were selected from Fayoum and Bani Suef governorates; respectively. The full genome of both isolates was deposited in GenBank under accession numbers listed in (Table 1).

Fig. 2: Gel electrophoresis of rolling circle amplification (RCA) followed by restriction fragment length polymorphism (RFLP) products using HpaII restriction enzyme. Samples 1–22 are squash samples collected from Fayoum and Bani Suef governorates; samples No 23 and 24 are healthy squash as negative controls. Expected fragments sizes (bp) obtained by in silico digestion of the retrieved sequences are shown in between the gels; DNA-A and DNA-B were denoted A (red) and B (blue) letters; respectively. Red asterisks refer to samples with diverse polymorphic RCA/RFLP pattern. M molecular weight marker.

Fig. 3 Gel electrophoresis of rolling circle amplification (RCA) followed by restriction fragment length polymorphism (RFLP) products using Sau3A1 restriction enzyme. Samples 5 and 8 that show different banding pattern from Bani Suef Governorate. Black brackets indicated the eluted fragments between 2.5 and 2.8 kb used in cloning. M molecular weight marker.


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The genome organization of both isolates, SLCV-Sq-EG1 and SLCV-Sq-EG2, showed the typical genome organization of new world bipartite begomoviruses. Five open reading frames (ORFs) on DNA-A component, AC1 ORF encoding replication protein (Rep), AC3 ORF encoding transcriptional activator protein (TrAP), AC2 ORF encoding replication enhancer protein (Ren), AC4, and coat protein (CP) (Lazarowitz, 1991). Two ORFs on DNA-B component, BC1 ORF encoding movement protein (MP) and BV1 ORF encoding nuclear shuttle protein (NSP). These proteins (MP and NSP) playing a key role in intracellular and cell to cell movement of the virus in plants (Lazarowitz et al., 1992).

SLCV Egyptian isolates, SLCV-Sq-EG1 and SLCV-Sq-EG2, shared 99% sequence identity between each other in DNA-A and DNA-B (Tables 1 and 2). Comparison analysis was carried out with (SLCV-Sq-EG1 and SLCV-Sq-EG2) and other begomoviruses available in GenBank. The highest nucleotide identity 99.1 and 98.3% in DNA-A and DNA-B, respectively, was detected with SLCV-Sq-PAL isolate infecting squash in Palestine (Tables 1 and 2). Whereas, the lowest nucleotide identity (91.5%) was found withSLCV-Sq-EG2 and SLCV-Cot-PAK isolate which infecting cotton in Pakistan (Table 2).

A more detailed analysis revealed that AV1 ORFs shared the highest amino acid identity (99.6 %) with those of SLCV-Sq-PAL, and SLCV- Sq-EG3. ORFs (AC1 and AC3) showed more than 97% amino acid identity with all SLCV isolates, except SLCV-To-EG4 which show the lowest sequence identity (89.6%) in AC1 ORFs (Tables 1 and 2). ORFs AC2 was highly diverse, it showed as low as 72.9% amino acid identity with SLCV-Sq-EG3. In DNA-B component, BV1 ORFs shared 97.8 % amino acid identity with SLCV-Sq-PAL found in squash in Palestine, while BC1 ORFs shared 99.3 %, amino acid identity with SLCV-Cot-PAK isolate affecting cotton in Pakistan (Tables1 and 2) Phylogenetic analysis

Phylogenetic analysis of SLCV-Sq-EG1 and SLCV-Sq-EG2 isolates were performed based on the alignment of nucleotide sequences of DNA-A and DNA-B with selected begomoviruses (Fig. 4). Both SLCV-Sq-EG1 and SLCV-Sq-EG2 isolates were clustered together with other SLCV isolates from Middle East countries (Egypt, Palestine, Jordan, Lebanon, Israel, and Oman) in one clade with high bootstrap value. Whereas, SLCV isolates from Pakistan and USA usually cluster together when DNA-A and DNA-B sequences used to construct the phylogenetic trees (Fig. 4).

Fig. 4: Phylogenetic analysis of Squash leaf curl virus (SLCV), based on a multiple sequence alignment of the complete DNA-A (A) and DNA-B (B) components of selected begomoviruses. SLCV-Sq-EG1 and SLCV-Sq-EG2, marked by black circles. Trees were constructed by the Neighbor-joining method. The trees were bootstrapped with 1000 replication using MEGA 7 software with values provided at the branch nodes. The scale bar represents the number of nucleotide substitution per site. Watermelon chlorotic stunt virus (WmCSV) was used as outgroups species. Acronyms of all isolates and accession numbers are provided in table 1 and 2.


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Table 1: Percentage nucleotide and amino acid sequence identities (%) between SLCV-Sq-EG1 and other SLCV isolates from different hosts and countries. The highest percentage of sequence identities is shaded.

SLCV Isolates Country Host Accession numbers Total nt. DNA-A DNA-B

DNA-A DNA-B DNA-A DNA-B AV1 AC1 AC2 AC3 AC4 BV1 BC1

SLCV-Sq-EG1 Egypt Squash MK284929 MK284930 ID ID ID ID ID ID ID ID ID

SLCV-Sq-EG2 Egypt Squash MK284931 MK284932 98.9 98.8 98.8 98.5 98.2 99.2 98.4 99.2 98.9

SLCV- Sq-EG3 Egypt Squash KC895398 KF030954 99.0 90.4 99.6 98.8 72.9 97.7 98.4 82.6 69.0

SLCV-To-EG4 Egypt Tomato MG763920 MG763921 95.6 95.0 95.6 89.6 95.4 97.7 77.6 82.3 84.6

SLCV-Pep-EG5 Egypt Pepper MH346454 MH346455 96.6 97.2 96.4 94.5 95.4 97.7 99.2 79.1 92.1

SLCV-Sq-OM Oman Squash HG969277 HG941652 99.0 98.2 99.2 99.4 98.8 99.2 98.4 89.3 99.6

SLCV-Sq-PAL Palestine Squash KC441465 KC441466 99.1 98.3 99.6 98.2 74.1 97.7 96.8 97.8 98.9

SLCV-To-JO Jordan Tomato JX444577 JX444574 99.0 98.2 98.8 99.1 98.8 98.5 99.2 86.2 98.9

SLCV-Sq-LB Lebanon Squash HM368373 HM368374 99.2 98.3 99.2 98.8 74.7 99.2 99.2 85.8 98.6

SLCV-Sq-IL Israel Squash KM595114 HQ184437 98.8 98.5 98.8 99.1 98.8 99.2 99.2 86.2 98.6

SLCV-Cot-PAK Pakistan Cotton MF504010 MF504013 91.3 96.4 86.4 97.1 74.1 97.0 94.4 96.1 99.3

SLCV-Sq-US USA Squash DQ285016 DQ285018 96.6 95.3 98.0 98.2 74.1 99.2 93.6 93.9 98.2

WmCSV-WM-PAL Palestine Watermelon KC462552 KC462553 56.4 41.6 70.9 51.6 35.5 50.3 8.8 25.6 40.4


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Table 2: Percentage nucleotide and amino acid sequence identities (%) between SLCV-Sq-EG2 and other SLCV isolates from different hosts and countries. The highest percentage of sequence identities is shaded.

SLCV Isolates Country Host Accession numbers Total nt. DNA-A DNA-B

DNA-A DNA-B DNA-A DNA-B AV1 AC1 AC2 AC3 AC4 BV1 BC1

SLCV-Sq-EG1 Egypt Squash MK284929 MK284930 98.9 98.8 98.8 98.5 98.2 99.2 98.4 99.2 98.9

SLCV-Sq-EG2 Egypt Squash MK284931 MK284932 ID ID ID ID ID ID ID ID ID

SLCV- Sq-EG3 Egypt Squash KC895398 KF030954 99.1 90.2 99.2 98.5 72.9 98.5 98.4 82.3 68.7

SLCV-To-EG4 Egypt Tomato MG763920 MG763921 95.7 94.8 95.2 89.3 95.9 98.5 76.8 81.9 84.3

SLCV-Pep-EG5 Egypt Pepper MH346454 MH346455 96.8 97.0 96.0 94.2 95.9 98.5 99.2 78.7 91.4

SLCV-Sq-OM Oman Squash HG969277 HG941652 99.1 98.1 98.8 99.1 99.4 100 98.4 89.3 99.3

SLCV-Sq-PAL Palestine Squash KC441465 KC441466 99.2 98.5 99.2 97.9 74.1 98.5 96.8 97.8 98.6

SLCV-To-JO Jordan Tomato JX444577 JX444574 99.2 98.2 98.4 98.8 99.4 99.2 99.2 85.8 98.6

SLCV-Sq-LB Lebanon Squash HM368373 HM368374 99.3 98.5 98.8 98.5 74.7 100 99.2 85.5 98.2

SLCV-Sq-IL Israel Squash KM595114 HQ184437 98.9 98.2 98.4 98.8 99.4 100 99.2 85.8 98.2

SLCV-Cot-PAK Pakistan Cotton MF504010 MF504013 91.5 96.2 86.4 96.8 74.1 97.7 94.4 96.1 98.9

SLCV-Sq-US USA Squash DQ285016 DQ285018 96.7 96.2 97.6 97.9 74.1 100 93.6 93.9 97.9

WmCSV-WM-PAL Palestine Watermelon KC462552 KC462553 56.4 41.6 70.5 51.3 35.5 50.3 8.8 25.6 40.4


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Recombination analysis We investigated the possibility of recombination events located within DNA-A and DNA-B of

SLCV isolates (SLCV-Sq-EG1 and SLCV-Sq-EG2) using RDP v.4 Software (Martin et al., 2015). Five recombination events were detected in the DNA-A and DNA-B components (Fig. 5; Table 3). In DNA-A, SLCV-Sq-EG1 and SLCV-Sq-EG2 are recombinants with the major parent being a SLCV-To-EG4 like isolates and the SLCV-Sq-PAL like isolates. SLCV-Pep-EG5 isolate is a recombinant with SLCV-WM-USA like isolates as the major parent and SLCV-To-EG4 infecting tomato in Egypt as a minor parent. SLCV-Cot-PAK isolate is the third recombinant detected in DNA-A component, with SLCV-Sq-PAL like isolates as the major parent and SLCV infecting tomato isolate from Egypt (SLCV-To-EG4) as a minor parent (Fig. 5; Table 3).

In DNA-B, two recombinants (4 and 5) were detected with strong statistical significant. Isolates SLCV-Sq-EG1 and EG2 are recombinants with SLCV isolate infecting squash from USA and SLCV isolate infecting squash in Egypt (SLCV-Sq-EG3) as minor and major parents; respectively.

Finally, recombination breakpoints were detected in DNA-B of SLCV isolate infecting tomato from Egypt (SLCV-To-EG4), with Jordanian SLCV isolate infecting tomato (SLCV-To-Jo) and Israeli SLCV isolates infecting squash (SLCV-Sq-IL) as minor and major parents; respectively. All recombinants were detected by at least six algorithms with strong statistical significance (Table 3).

Table 3: List of SLCV isolates showing evidence of putative recombination events in DNA-A and

DNA-B. The analysis performed using RDP v.4 software.

SLCV Recombinant Breakpoint Positions

RDP Methodsa ( P-value)

Start End R G B M C S 3S

DNA-A 1-SLCV-Sq-EG1&2 974 1205 3.4E-2 3.56E-2 1.4E-2 1.5E-2 2.89E-2 7.1E-3 1.43E-2

2- SLCV-Pep-EG5 960 1389 3.43E-02 3.56E-03 6.01E-03 9.87E-03 2.53E-02 7.79E-08 5.43E-03

3- SLCV-Cot-PAK 207 2132 8.40E-13 8.81E-12 3.10E-05 1.55E-14 9.60E-15 1.38E-32 5.77E-32

DNA-B 4-SLCV-Sq-EG1&2 415 612 1.02E-06 4.13E-06 8.74E-04 1.49E-04 6.57E-05 6.04E-09 1.48E-06

5- SLCV-To-EG4 1661 2011 4.91E-07 1.21E-06 7.27E-08 1.43E-09 3.23E-09 5.38E-12 1.96E-10 aAlgorithms implemented in RDP v.4 software were; R, RDP; G, GeneConv; B, Bootscan; M, MaxChi; C, CHIMERA; S, SiScan; 3S, 3SEQ. The support probability for each algorithm is shown.

Fig. 5. Schematic representation of the possible recombination events in the DNA-A and DNA-B of SLCV isolates using recombination detection program (RDP v.4). Events 1-3 in DNA-A and events 4-5 in DNA-B. A linear genome map of begomoviruses is plotted on top of the figure, with the position of genes and their orientation indicated by arrows to show the relative position of recombinant breaking points.


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Discussion Symptoms of SLCD on squash plants found in Fayoum and Bani Suef governorates, the

collection sites for this study. Feld survey, revealed the high incidence of begomovirus-like symptoms, is in line with the previous studies in Egypt, where begomoviruses were detected in a majority of tested squash plants using degenerate primers (Abdel-Salam et al., 2006; Awad et al., 2018; Idris et al., 2006; El-Dougdoug et al., 2014). Amplification of all circular DNA forms using RCA combining with RLFP (RCA/RFLP), including viral DNA molecules and presumably cognate satellite DNAs, making this technique fast, reliable and cheap when testing for circular DNA viruses without any previous sequence information compared to PCR (Inoue-Nagata et al., 2004; Jeske et al., 2014; Kushawaha et al., 2018; Laney et al., 2012; Paprotka et al., 2010; Wyant et al., 2011).

Symptomatic squash samples collected from different localities in Egypt were tested for the possible presence of begomoviruses, using RCA/RFLP. All PCR-positive samples tested positive using RCA/RFLP produced eight bands after RCA products digested with the HpaII restriction enzyme, in line with previous study (Ali-Shtayeh et al., 2014). The sum of the fragments obtained by (RFLP) was at least 5 kb, which is consistent with an expected result from sequencing results. No prove of mixed infection of SLCV with WmCSV or TYLCV, albite the limited number of tested samples, this agreed with the earlier reports of the survey performed in Egypt and nearby countries (Lapidot et al., 2014).

RCA/RFLP has been proven to facilitate simultaneous detection of multiple begomoviruses and cognate satellites in one reaction, (Haible et al., 2006; Inoue-Nagata et al., 2004; Jeske, 2018; Paprotka et al., 2010; Schubert et al., 2007; Wyant et al., 2011). Sequence analysis of two different isolates from Fayoum and Bani Suef, showed high sequence identity (96-99%) with known SLCV sequences in GenBank, indicating them to be variants of SLCV.

Applying partial digestion by Sau3A1 restriction enzyme of RCA products followed by cloning strategy using BamHI-cut plasmid, proved to be a useful approach to get the full genome sequences, without prior knowledge on virus sequence understudy (Jeske, 2018; Wyant et al., 2011). In addition, RCA/RFLP technique greatly facilitate simultaneous detection of mixed viral infection and identification of the respective begomoviruses and cognate satellites in the same sample (Jeske et al., 2014; Paprotka et al., 2010).

In RCA/RFLP analysis, the explanation for the presence of a different pattern (samples No. 5 and 8) (Fig. 2), most likely due to the point mutations in the viral DNA due to polymorphism within the viral quasi-species (Inoue-Nagata et al., 2004; Jeske, 2018; Paprotka et al., 2010; Schubert et al., 2007; Wyant et al., 2011). These mutations could lead to changes in the recognition sites for HpaII restriction enzyme (Haible et al., 2006; Jeske et al., 2010; Schubert et al., 2007). Non-assigned small fragments (sample No. 1; Fig. 2) which may result from small circular DNAs from the plant, like mitochondrial DNA, however this a few unexpected bands not affecting the specificity and sensitivity of the RCA/RFLP technique (Homs et al., 2008; Jeske et al., 2010; Wyant et al., 2015).

Phylogenetic analysis of SLCV isolates showed low geographical structure distribution, probably attributed to a founder effect, where a population established recently in Middle Eastern countries (Abdel-Salam et al., 2006; Abudy et al., 2010; Ali-Shtayeh et al., 2014; Al-Musa et al., 2008; Awad et al., 2018; Lapidot et al., 2014). This observation supports the notion that a single introduction of SLCV into the region, with limited time for diversification (Lapidot et al., 2014). There are accumulative evidence suggests that begomoviruses are prone to recombination (Albuquerque et al., 2013, 2012; Al-Saleh et al., 2014; Bull et al., 2007; De Bruyn et al., 2016; Martin et al., 2011; Padidam et al., 1999; Serfraz et al., 2015). Recombination is considered as a powerful driving force of genetic diversity and adaptation of begomoviruses, thereby potentially increasing their genetic fitness and host range (Belabess et al., 2016; Navas-Castillo et al., 2000; Prasanna and Rai, 2007; Rojas et al., 2005; Serfraz et al., 2015). In the present study, analysis has shown that recombination occurs in DNA-A and DNA-B of SLCV isolates not rare, in line with the previous study (Lapidot et al., 2014). Interestingly, most recombination events were detected in the SLCV isolates infecting hosts other than cucurbits such as cotton, pepper, tomato, and watermelon. In conclusion, these results are indicative that recombination in SLCV could play a role in expanding the virus host range. Similar to other begomoviruses, recombination providing additional sources of variation in SLCV Egyptian isolates with unpredictable effects on virus-host adaptation. Also, we propose RCA/RFLP as a reliable diagnostic technique to scrutinize for begomoviruses infection, despite any prior knowledge about viral sequences.


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Acknowledgement This work was partially supported by grant from Science and Technology Development Fund

(STDF project number 15067).

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Rolling circle amplification-based detection and ... · Rahmany, 2015; Farrag et al., 2014; Lapidot et al., 2014). These techniques require a prior knowledge on nucleotide sequences