Charlock mustard (Sinapis arvensis): a weed reservoir for begomoviruses and associated betasatellite in Jordan

Abstract

Four crop-infecting begomoviruses were found naturally affecting Charlock mustard (Sinapis arvensis L.) plants in Jordan and characterized at the molecular level. Symptoms included leaf curling and stunting. PCR analysis showed the presence of Tomato yellow leaf curl virus (TYLCV), Tomato yellow leaf curl Sardinia virus (TYLCSV), Squash leaf curl virus (SLCV) and Watermelon chlorotic stunt virus (WmCSV). These viruses were associated with Cotton leaf curl Gezira betasatellite (CLCuGβ). Sequence analysis revealed that TYLCV and TYLCSV had 98.5% and 99.3% nucleotide (nt) sequence identities with a previously described TYLCV strain from Florida and TYLCSV from Sardinia, respectively. The other two viruses, SLCV and WmCSV, shared 99.7% and 99.5% nt identities with previously described strains from Jordan. Sequence analysis also showed that CLCuGβ had 98.8% nt identity with a strain from Israel. To our knowledge, this is the first report of S. arvensis as a natural host of SLCV, WmCSV, TYLCSV and CLCuGβ.

Résumé

On a découvert que quatre bégomovirus s’attaquant aux cultures infectaient naturellement la moutarde sauvage (Sinapis arvensis L.) en Jordanie; ceux-ci ont été caractérisés à l’échelle moléculaire. Les symptômes incluaient l’enroulement des feuilles et le rabougrissement des plants. L’analyse par PCR a révélé la présence du virus de la jaunisse de la tomate (TYLCV), du virus sarde de la jaunisse de la tomate (TYLCSV), du virus de l’enroulement des feuilles de la courge (SLCV) et du virus du rabougrissement chlorotique de la pastèque (WmCSV). Ces virus étaient associés au bêta-satellite du virus de l’enroulement des feuilles du coton de la Gezira (CLCuGβ). L’analyse de séquences a révélé que le TYLCV et le TYLCSV affichaient des identités de séquences nucléotidiques (nt) de 98.5% et de 99.3%, respectivement, avec une souche floridienne de TYLCV et une sarde de TYLCSV antérieurement décrites. Les deux autres virus, SLCV et WmCSV, partageaient des identités de séquences de 99.7% et de 99.5% avec des souches jordaniennes décrites antérieurement. L’analyse de séquences a également révélé que le bêta-satellite CLCuGβ affichait une identité de séquences de 98.8% avec une souche israélienne. À notre connaissance, c’est la première fois que l’on mentionne que S. arvensis sert d’hôte naturel au SLCV, au WmCSV, au TYLCSV et au CLCuGβ.

Keywords:

• begomoviruses
• betasatellite
• Brassicaceae
• virus detection
• weed hosts

Mots clés:

• adventices hôtes
• bégomovirus
• bêta-satellite
• Brassicaceae
• détection des virus

Introduction

Geminiviruses are a large group of plant viruses that cause several important crop diseases worldwide. Begomovirus, the largest and most economically important genus in the Geminiviridae family, encompasses viruses that are transmitted by the whitefly Bemisia tabaci (Gennadius) and infect only dicotyledonous plants (Harrison & Robinson 1999). Begomoviruses can either have monopartite or bipartite genomes. Bipartite genomes consist of two ssDNA molecules of approximately 2.7 kb size (Brown et al. 2012). Most of the monopartite begomoviruses are associated with a circular DNA molecule of ~1.4 kb referred to as betasatellites (Briddon et al. 2003). The betasatellites depend on their helper begomoviruses for replication, and many betasatellites are essential for the induction of typical disease symptoms and viral genome accumulation (Zhou 2013).

Until the last decade, information on the occurrence of begomoviruses in the eastern Mediterranean basin was limited to Tomato yellow leaf curl virus (TYLCV). In 2000, Squash leaf curl virus (SLCV), which was reported for the first time in California, USA (Flock & Mayhew 1981) has been detected in Saudi Arabia (Al-Shahwan et al. 2002); since then, the virus has spread throughout the region (Antignus et al. 2003; Lapidot et al. 2014). The virus was found predominantly affecting squash crops in Jordan, Israel, Egypt, Lebanon and Palestine, causing leaf mottling, curling and severe stunting of affected plants (Idris et al. 2006; Al-Musa et al. 2008; Abudy et al. 2010; Ali-Shtayeh et al. 2012; Sobh et al. 2012). Watermelon chlorotic stunt virus (WmCSV) is another newly introduced begomovirus to the region (Abudy et al. 2010; Al-Musa et al. 2011; Samsatly et al. 2012). The virus was first reported in Yemen infecting nearly all cultivated cucurbits and causes severe damage mainly to watermelon (Citrullus lanatus L.) and melon (Walkey et al. 1990).

Previously, we reported the association of SLCV and WmCSV with tomato yellow leaf curl disease in tomato crops in Jordan (Haj Ahmad et al. 2013). These two viruses pose a major threat to vegetable cultivation throughout the Mediterranean basin. Most previous studies have focused on begomoviruses that are affecting crop plants; however, these viruses are also able to infect a variety of weed species which serve as important alternative or overwintering hosts. In the last few years, increasing number of reports have shown that weeds act as natural reservoirs for many begomoviruses (Hernández-Zepeda et al. 2010; Mubin et al. 2010; Papayiannis et al. 2011; Silva et al. 2012; Kil et al. 2014; De Bruyn et al. 2015; Tahir et al. 2015; Zaidi et al. 2016). In addition, mixed begomovirus infections and recombination were reported in many crops and weeds (Sanz et al. 2000; Mansoor et al. 2003; Jovel et al. 2004; Morilla et al. 2004; Castillo-Urquiza et al. 2008).

In this study, we characterized four crop-infecting begomoviruses and associated betasatellite from naturally infected Charlock mustard (Sinapis arvensis L.) plants exhibiting severe leaf curling and stunting symptoms. The identities of the virus strains were determined following PCR and phylogenetic analysis.

Materials and methods

Virus source

In 2011, leaf tissues were collected from six symptomatic S. arvensis plants found at Amman National Park, Jordan. Plants showed typical disease symptoms caused by begomoviruses, including severe leaf curling and general stunting (Fig. 1). Leaf tissues were kept at −20°C until analysis.

Fig. 1 (Colour online) A plant of Charlock mustard (Sinapis arvensis) showing severe leaf curling symptoms due to natural infection by a complex of begomoviruses.

DNA extraction and PCR amplification

Total nucleic acids were extracted from all six S. arvensis plants as follows: leaf tissues (200 mg) were ground in liquid nitrogen and 600 μL of the extraction buffer (100 mM Tris–HCl, pH 8.0, 50 mM EDTA, 500 mM NaCl, 10 mM 2-mercaptothanol), and 1% SDS were added to the extract. The mixture was incubated at 65°C for 10 min and then 200 μL of 5 M potassium acetate was added. Tubes were kept on ice for 10 min and centrifuged at 1400 rpm for 10 min at 4°C. Two volumes of 95% ethanol were added to the supernatant and after centrifugation, pellets were washed with 70% ethanol and resuspended in 50 μL of nuclease-free water. To detect begomoviruses and associated betasatellite, total nucleic acids extracted from the six symptomatic plants were used as template in PCR using virus-specific primers (Supplementary Table 1). For example, the primer pairs TYv2337/TYc138 and TYAlmv2516/TYAlmc115 were used to detect TYLCV and Tomato yellow leaf curl Sardinia virus (TYLCSV), respectively, while SLCV and WmCSV were detected using primer pairs SLCVNeF/SLCVNeR and WmCSVNeF/WmCSVNeR, respectively (Supplementary Table 1). The betasatellite was detected using the primer pair β01/β02 (Briddon et al. 2002).

Table 1. Genome characteristics of begomoviruses reported in this study.

Virus	DNA-A	DNA-B	AV1 (nt/aa)	AV2 (nt/aa)	AC1 (nt/aa)	AC2 (nt/aa)	AC3 (nt/aa)	AC4 (nt/aa)	BV1 (nt/aa)	BC1 (nt/aa)
TYLCV-[JO:Sin:11]	2771	–	777/258	–	1074/357	408/135	405/134	294/97	–	–
TYLCSV-[JO:Sin:11]	2776	–	777/258	243/80	1080/359	408/135	405/134	297/89	–	–
WmCSV-[JO:Sin:11]	2753	2772	777/258	360/119	1086/361	408/135	405/134	144/47	765/254	888/295
SLCV-[JO:Sin:11]	2634	2619	756/251	–	1044/347	525/174	405/134	378/125	576/191	882/293

Amplification, cloning, sequencing and phylogenetic analyses of full-length genomes

Results of PCR analysis showed that the six S. arvesis plants were infected with TYLCV, TYLCSV, SLCV, WmCSV and a betasatellite; therefore, total nucleic acids extracted from one symptomatic S. arvesis plant was used to amplify circular DNAs using Phi-29 polymerase, essentially according to the manufacturer’s instructions (Amersham Biosciences, Piscataway, NJ, USA). After that, amplified circular DNAs were used as template in PCR to amplify the full-length genomes of begomoviruses using virus-specific primers (Supplementary Table 1). For instance, BT18-F1/BT18-R1 and BT18-F2/BT18-R2 primer pairs were used to amplify TYLCV full-genome, while the full-genome of TYLCSV was amplified using the primer pairs BT27-F1/BT27-R1 and BT27-F2/BT27-R2. DNA-A and DNA-B of SLCV were amplified using primer pairs SLCVF-SalI/SLCVR-SalI and SLCVBIF/SLCVBIR, respectively. DNA-A of WmCSV was amplified using the primer pairs WF2F1/WAI-XbaIc and W4146F2/W4146R2, while DNA-B was amplified using WAI-SmaIv/WAI-SmaIc primer pairs. The betasatellite molecule was amplified using the β01/β02 primers.

PCR products were gel-eluted and cloned into pGEM T-Easy vector (Promega, USA) according to the manufacturer’s instructions. Recombinant plasmids containing full-length inserts were sequenced by primer walking (Macrogen Inc.) and consensus sequence data obtained were deposited in GenBank database. Sequences were aligned with sequences of selected begomoviruses available in GenBank (Supplementary Table 2) using CLC main workbench 5.6 software (CLC bio A/S, Denmark). ORF finder (http://www.ncbi.nlm.nih.gov/projects/gorf/) was used to identify ORFs and all pairwise comparisons were performed using the MUSCLE algorithm (Edgar 2004) implemented in the Species Demarcation Tool (SDT) version1.2 (Muhire et al. 2014). MEGA 6.06 (Tamura et al. 2013) was used to generate phylogenetic trees using the maximum likelihood method and 1000 bootstrap replicates to assess branch support. The detection of potential recombination events in the begomovirus sequences, including identification of likely parental sequences and localization of possible recombination breakpoints, was carried out using the Recombination Detection Program (RDP3) with default settings (Martin et al. 2010).

Results

Detection and sequence analysis of begomoviruses and a betasatellite

To investigate the association of begomoviruses with the disease symptoms observed on S. arvensis, PCR was conducted using virus-specific primer pairs. The expected amplicon sizes of TYLCV (~630 bp), TYLCSV (~430 bp), SLCV (~380 bp), WmCSV (~440 bp) and betasatellite (~1300 bp) could be detected in DNA extracted from all six S. arvensis plants, indicating the association of these viruses with the leaf curling and severe stunting symptoms. Since PCR data showed that all tested plants were infected with the four begomovirus and the betasatellite, DNA extracted from one plant was further used to amplify the full-length genomes using specific primers. Amplicons were cloned, sequenced and submitted to GenBank. Sequence analysis revealed that the genomes of TYLCV, hereafter Tomato yellow leaf curl virus-[Jordan:Sinapis:2011] (TYLCV-[JO:Sin:11]; JX131286), and of TYLCSV hereafter Tomato yellow leaf curl Sardinia virus-[Jordan:Sinapis:2011] (TYLCSV-[JO:Sin:11]; JX131285), were 2771 bp and 2776 bp in length, respectively. Whereas, the length of DNA-A (JX131281) and DNA-B (JX131282) of SLCV, hereafter Squash leaf curl virus-[Jordan:Sinapis:2011] (SLCV-[JO:Sin:11]), were 2634 bp and 2619 bp, respectively. The DNA-A (JX131283) and DNA-B (JX131284) of WmCSV, hereafter Watermelon chlorotic stunt virus-[Jordan:Sinapis:2011] (WmCSV-[JO:Sin:11]), had genomes length of 2753 bp and 2772 bp, respectively, and the full-length sequence of the betasatellite (JX649952) was determined to be 1339 bp. Sequences were initially analysed by BLAST, confirming the successful cloning of putative full-length begomovirus genome.

Sequences of cloned genomes showed organizations typical of begomoviruses i.e. DNA-A components with one or two open reading frames on the viral-sense strand (AV2/AV1) and four ORFs on the complementary-sense strand (AC1, AC2, AC3 and AC4) and DNA-B components with one ORF on the viral-sense strand (BV1) and one ORF on the complementary-sense strand (BC1). A summary of the virus genome features is presented in Table 1. SDTv1.2 analysis revealed that both DNA-A (Fig. 2a) and DNA-B (not shown) of SLCV-[JO:Sin:11] had the highest nt sequence identity of 99.7% and 98.8%, respectively, to the Jordanian isolate SLCV-[JO2-23] (KM595177). The iteron TGGTGTCC was identified in SLCV-[JO:Sin:11] with two direct repeat copies (nt 2509–2516, 2547–2554) and two inverted repeat copies GGACACCA (nt 2509–2516 and 2596–2603). The corresponding iteron-related domain, SFRLT, was identified in the Rep protein.

Fig. 2 (Colour online) Colour-coded pairwise identity matrix generated from 23 complete DNA-A genome segment of begomoviruses. The analyses include SLCV-[JO:Sin:11] (a) and TYLCSV-[JO:Sin:11] (b) from this study (in red) and the corresponding published sequences of selected begomoviruses. The matrix was generated using the program Sequence Demarcation Tool (SDT v.1.2; http://web.cbio.uct.ac.za/), and each coloured cell represents a percentage identity score for two sequences. Coloured keys indicating the correspondence between pairwise identities and the colours displayed in the matrix are presented.

Comparison of the nt sequences of the ORFs of SLCV-[JO:Sin:11] DNA-A with those of the corresponding ORFs of closely related SLCV isolates showed that both AV1 and AC1 shared highest nt (100% and 99.3%) and aa (100% and 99.0%) similarities, respectively, with SLCV-[JO2-23] (Supplementary Table 3). However, AC2 and AC3 had 100% nt and aa similarities with the Israeli isolate SLCV-[IL:IsSq3] (KT099131) and SLCV-[JO2-23] (KM595177), respectively. The homology inferred from the sequence similarity was supported by a phylogenetic analysis using Maximum likelihood method, in which SLCV-[JO:Sin:11] grouped in the clade containing SLCV isolates from Jordan and Palestine (Fig. 3).

Fig. 3 Maximum likelihood phylogenetic tree based on the complete DNA-A sequence of TYLCSV-[JO:Sin:11], TYLCV-[JO:Sin:11], WmCSV-[JO:Sin:11] and SLCV-[JO:Sin:11] (arrows) and sequences of selected begomoviruses available in GenBank. Support for nodes in a bootstrap analysis with 1000 replications is shown. The tree was generated using the Maximum likelihood method in MEGA 6.06. An isolate of Beet curly top virus (BCTV-[US:Sp3]) AY548948 was included as outgroup.

The heat map generated with the SDTv1.2 showed that the DNA-A of WmCSV-[JO:Sin:11] shared 99.5% nt identity with WmCSV-[JO:JO3-627] (KM820233), whereas the DNA-B component had 97.1% nt similarity with that of WmCSV-[JO:JOR] (EU561237) (data not shown). The iteron ATTGG was identified in WmCSV-[JO:Sin:11] with two direct repeat copies (nt 2639–2643, 2674–2678). The IRD, FRIQ, was identified in the Rep protein. The sequences of AV1 and AV2 ORFs of WmCSV-[JO:Sin:11] were compared with published sequences of other isolates (Supplementary Table 4). The nt and aa sequences of these two ORFs were found to be identical to that of WmCSV-[JO:JOR] (EU561237), whereas AC1 showed 99.5% similarity at nt and aa levels to that of WmCSV-[JO:JO3-627] (KM820233). AC4 ORF was found identical (100%) at the nt and aa levels to that of all tested WmCSV isolates. Phylogenetic analyses on the basis of alignment of complete nucleotide sequences placed WmCSV-[JO:Sin:11] in a clade containing other isolates from Jordan, Lebanon and Israel (Fig. 3).

When the sequence of TYLCV-[JO:Sin:11] was compared with that of closely related isolates, the percentage of nt identity ranged from 97.1% to 98.5% (Supplementary Table 5). Sequence comparison showed that TYLCV-[JO:Sin:11] shared the highest (98.5%) nt sequence identity with TYLCV-[US:FL] (AY530931) from Florida, USA. A phylogenetic tree based on the alignment of TYLCV-[JO:Sin:11] and other selected begomovirus sequences was constructed with the Maximum likelihood method of the MEGA6.0. As demonstrated in Fig. 3, TYLCV-[JO:Sin:11] formed one cluster with other TYLCV isolates from the New World. When individually encoded proteins were compared, the V1 ORF of TYLCV-[JO:Sin:11] showed maximum aa identity (99.6%) with TYLCV-[US:FL] (AY530931), TYLCV-[DO] (AF024715) and TYLCV-[CU:C2+:11] (KM926626) isolates. However, V2 ORF of the virus could not be identified due to a thymine deletion at position 137, introducing a frame shift (data not shown). In the case of the C1-encoded Rep protein, it showed maximum aa (98.3%) identity with isolate TYLCV-[US:FL], while C2 ORF had the highest level of nt identity (99.0%) with the Jordanian isolate TYLCV-[JO:Ju:08] (GQ861426). Regarding C3 ORF, it showed 98.0% nt and 95.6% aa identity with TYLCV-[JO:Ju:08] and C4-encoded protein had maximum aa identity (99.0%) with TYLCV-[DO]. The iteron AATCGGTGTC was identified in TYLCV-[JO:Sin:11] at nt 2622–2631 and the IRD, MPRLFKIY, was identified in the Rep protein.

Results of the pairwise sequence analysis of TYLCSV-[JO:Sin:11] (Fig. 2b) indicated that TYLCSV-[JO:Sin:11] is closely related to TYLCSV-[IT:Sar:88]. They shared 99.3% nt identity (Supplementary Table 6). When the deduced aa sequences were compared, ORFs of TYLCSV-[JO:Sin:11] showed the highest sequence identities with counterparts of TYLCSV-[IT:Sar:88]. The phylogenetic tree based on comparison of the full-length genome sequences showed that TYLCSV-[JO:Sin:11] is similar to TYLCSV-[IT:Sar:88] and clustered with other isolates from other Mediterranean countries (Fig. 3). The iteron GGGGG was identified in TYLCSV-[JO:Sin:11] at nt 2630–2638 and the IRD, GRFSIK, was identified in the Rep protein.

The sequence of the betasatellite detected in this study showed the typical arrangement of other betasatellites, with a single ORF of 354 nt in length (coordinates 549–196 nt) on the complementary-sense strand encoding BC1 protein, an A-rich region of ~240 nt and a satellite conserved region (SCR) of ~220 nt in which the nonanucleotide TAATATTAC along with GCTCGCCCACG and CGTGGGCGAGC formed the putative stem and loop structure (Briddon et al. 2003). Sequence comparison with other betasatellites available in GenBank using SDTv1.2 revealed that the betasatellite, hereafter called Cotton leaf curl Gezira betasatellite-[Jordan:Sinapis:2011] (CLCuGβ-[JO:Sin:11]), shared maximum nt identity of 98.8% with that of CLCuGβ-[IS:IsSq1:11] (KU095847) (data not shown), recently reported in Israel (Rosario et al. 2016). When the aa sequence of the BC1 ORF was compared, it showed the highest (98.0%) identity with that of CLCuGβ-[IS:IsSq1:11]. Phylogenetic analysis revealed that CLCuGβ-[JO:Sin:11] clustered with other isolates of CLCuGβ previously reported from Egypt, Israel and Jordan (not shown).

Recombination analysis

To detect putative recombinants among begomoviruses and betasatellite detected in this study, the full-length genomes were aligned together with those of selected begomoviruses and analysed using RDP3. A large recombination event with a degree of confidence by five recombination detection methods implemented in RDP3 (Bootscan = 5.81 × 10⁻⁰³, MaxChi = 5.134 × 10⁻⁰⁴, Chimaera = 1.731 × 10⁻⁰², SiScan = 3.205 × 10⁻⁰⁴, 3Seq = 4.693 × 10⁻⁰⁵) was detected in the DNA-A of WmCSV-[JO:Sin:11]. The recombination fragment involving WmCSV-[LB:LB1:09] (HM368371) as a major parent and WmCSV-[JO:JOR] (EU561237) as a minor parent is 1816 nt in size and located in the IR, AV2, AV1, AC2 and AC3 (breakpoint coordinates 2684–1746). No recombination events could be detected in the genomes of TYLCV-[JO:Sin:11], TYLCSV-[JO:Sin:11], SLCV-[JO:Sin:11] and CLCuGβ-[JO:Sin:11].

Discussion

Begomovirus diseases are a serious problem on many cultivated plants worldwide, especially in tropical and subtropical regions. Increasing numbers of reports demonstrate the importance of weeds as natural reservoirs of crop-infecting begomoviruses and their satellite molecules. For example, henbit deadnettle (Lamium amplexicaule) has recently been identified in Korea as a natural host for TYLCV (Kil et al. 2014). In addition, zinnia (Zinnia elegans), Asian copperleaf (Acalypha australis), cotton (Gossypium hirsutum), Velvetleaf (Abutilon theophrasti) and tobacco (Nicotiana tabacum) were found infected with TYLCV in eastern China (Li et al. 2014). Furthermore, weeds of the genera Ageratum, Asystasia, Clerodendrum, Emilia and Malvastrum were reported as reservoirs of begomoviruses infecting many crop species in west and central Africa (Leke et al. 2015).

In Jordan, reports of weeds that serve as natural hosts of begomoviruses are limited. In 1988, Siberian swallow-wart (Cynanchum acutum) and cheese weed (M. parviflora), collected from Jordan Valley, were found infected with TYLCV (Cohen et al. 1988) and 20 years later, we were able to characterize SLCV in M. parviflora plants collected from Jordan Valley with severe leaf curling symptoms (Al-Musa et al. 2008). Sinapis arvensis is one of the most widespread and abundant weeds in Jordan and many other neighbouring countries. Previous studies showed that S. arvensis is naturally infected with many viruses, including TYLCV, Cauliflower mosaic virus, Turnip mosaic virus, Sweet potato feathery mottle virus, Cucumber mosaic virus and Chickpea chlorotic stunt virus (Dikova 2008; Asaadet al. 2009; Akelet al. 2010; Papayianniset al. 2011). In this study, we were able to detect and characterize four crop-infecting begomoviruses and a betasatellite in S. arvensis. To our knowledge, this is the first report of S. arvensis as a natural host of SLCV, WmCSV, TYLCSV and CLCuGβ. The location from which the plants were collected, Amman National Park, is 20 km away from agricultural fields cropped to okra, squash and tomato. Begomoviruses reported in this study could be transmitted from infected plants in these fields to S. arvensis by viruliferous whiteflies. In Cyprus, Papayiannis et al. (2011) showed that TYLCV could infect S. arvensis in nature. The importance of these findings should be pointed out to farmers in order to adapt effective weed control measures in their fields. It is interesting to note that ORFs AC2 and AC3 of SLCV-[JO:Sin:11] and CLCuGβ-[JO:Sin:11] showed the highest nt similarities with the Israeli isolate SLCV-[IL:IsSq3] and CLCuGβ-[IS:IsSq1:11], respectively, which were recently identified in the whitefly B. tabaci collected from tomato and squash plants in Israel (Rosario et al. 2016). This is in accordance with our previous findings that tomatoes are naturally infected with SLCV, WmCSV and CLCuGβ-[JO:Hom:13] in Jordan (Haj Ahmad et al. 2013; Anfoka et al. 2014). It is worth mentioning that although V2 ORF, that encodes a protein essential for virus movement, could not be identified in the genome of TYLCV-[JO:Sin:11], the virus could be detected in newly emerged leaves of S. arvensis. Similar results were reported by Hak et al. (2015). They demonstrated that a mutant TYLCV carrying a non-translatable V2 could spread in Jimson weed plants at the same rate as the wild-type virus. These data together suggest that the V2 protein is not necessary for TYLCV movement in plants.

Sequence analysis of begomoviruses reported in this study demonstrated that these viruses belong to virus species and strains previously described in Mediterranean countries. An exception is TYLCV-[JO:Sin:11], which is closely related to other TYLCV isolates reported from the New World. This may indicate that TYLCV-[JO:Sin:11] was introduced into Jordan via long distance transport of infected plants or seeds. This is in accordance with results of a recent study that showed 100% transmission of TYLCV in tomato seeds (Kil et al. 2016).

Betasatellites show host specificity and can be divided into two major groups; one infecting malvaceous hosts and the other infecting non-malvaceous hosts (Briddon et al. 2003). Cotton leaf curl Gezira virus (CLCuGeV) is a geographically widespread begomovirus species occurring from central Africa to Jordan and infects a number of plant species, including cotton, okra, hollyhock and Sida spp. (Tahir et al. 2011). In 2009, we detected CLCuGeV for the first time in Jordan affecting hollyhock plants; however, all attempts to detect betasatellites in infected hollyhock plants failed. In this study, we report for the first time that S. arvensis acts as a natural reservoir of CLCuGβ. CLCuGβ-[JO:Sin:11] grouped together with other isolates previously reported from Jordan (CLCuGβ-[JO:Hom:13]) and neighbouring countries. CLCuGβ-[JO:Sin:11] showed maximum nucleotide sequence identity of 98.8% to CLCuGβ-[IS:IsSq1:11], previously identified from whiteflies in Israel (Rosario et al. 2016). Based on these findings, we propose that CLCuGβ-[JO:Sin:11] is an isolate of CLCuGβ-[IS:IsSq1:11] according to the recently updated ICTV begomovirus species demarcation criteria (Brown et al. 2015).

Betasatellites can be trans-replicated by geographically divergent and biologically diverse begomoviruses. In this study, CLCuGβ-[JO:Sin:11] has been detected in plants infected with divergent begomoviruses, suggesting that CLCuGβ-[JO:Sin:11] trans-replicated in S. arvensis by the noncognate helper begomoviruses. This is in accordance with results of previous studies where African cassava mosaic virus and BCTV could trans-replicate a defective molecule associated with TYLCV (Dry et al. 1997). In addition, TYLCV has been reported to trans-replicate distinct betasatellites from Japan and the Philippines (Ito et al. 2009). These data together demonstrate that S. arvensis can serve as a natural reservoir and a potential alternative host for crop-infecting begomoviruses and their associated betasatellites.

Supplemental material

Supplemental data for this article can be accessed online here: https://doi.org/10.1080/07060661.2017.1354332.

Additional information

Funding

This work was supported in part by USAID-MERC [grant number M26-063].