Sesbania bispinosa, a new host of a begomovirus-betasatellite complex in Pakistan

Abstract

Symptoms of severe leaf curling, yellowing and vein thickening, typical of begomovirus infection, were observed on Sesbania bispinosa grown in cotton leaf curl disease affected cotton fields in Pakistan. A begomovirus and its associated betasatellite were amplified and sequenced from these affected plants. Complete nucleotide sequence showed that a monopartite begomovirus named Pedilanthus leaf curl virus (PeLCV) is associated with the disease and it shared highest sequence identity (98.73%) with a clone previously reported from soybean. Characterization of the betasatellite showed that it had highest sequence identity (96–97%) with Tobacco leaf curl betasatellite (TbLCB), previously isolated from soybean. Our data shows the expansion of the host range of begomovirus-betasatellite complexes. To the best of our knowledge, this is the first occurrence of PeLCV and TbLCB on S. bispinosa.

Résumé

Les symptômes d’une grave frisolée, le jaunissement et l’épaississement des nervures, typiques de l’infection causée par les bégomovirus, ont été observés sur Sesbania bispinosa cultivé au Pakistan, dans des champs touchés par la frisolée du coton. Un bégomovirus et le satellite beta qui lui est associé, provenant de ces plants malades, ont été amplifiés et séquencés. La séquence entière des nucléotides a montré qu’un bégomovirus monopartite, nommé virus de la frisolée du pédilanthe (VFPe), est associé à la maladie et qu’il partage l’identité de séquence la plus élevée (98.73 %) avec un clone trouvé précédemment chez le soya. La caractérisation du satellite beta a montré qu’il partage l’identité de séquence la plus élevée (96–97 %) avec le satellite beta de la frisolée du tabac (BFTb), prélevé précédemment sur du soya. Nos données montrent l’expansion de la gamme d’hôtes des complexes bégomovirus-satellites beta. À notre connaissance, il s’agit de la première occurrence de VFPe et de BFTb sur S. bispinosa.

Keywords:

• Begomovirus
• betasatellite
• geminivirus
• Pedilanthus leaf curl virus
• Sesbania bispinosa

Mots clés:

• Begomovirus
• géminivirus
• satellite beta
• Sesbania bispinosa
• virus de la frisolée du pédilanthe

Introduction

Geminiviruses (family Geminiviridae) have received considerable attention among molecular biologists in the agriculture sector because of their increasing host range and severity of losses they cause to economically important crops (Moffat, 1999). While a lot of information is available on host-virus interactions in agricultural crops, comparatively less attention has been paid to the viruses that infect weeds and wild plant species, the potential reservoirs of viruses. During the off-seasons, most of the plant viruses remain latent in these alternate hosts and act as a key source for infecting economically important hosts during the cropping season. Sesbania bispinosa (family Fabaceae) is an important weed of fibre crops and legumes. During a routine survey in October 2013, leaves of several plants of S. bispinosa were found with severe leaf curling, yellowing and vein thickening symptoms (Fig. 1) in an infected cotton field in Bahawalpur district of Punjab province, Pakistan. Because of the typical symptoms, a begomovirus infection was suspected in these plants. The objective of this study was to characterize the potential virus infecting these plants.

Fig. 1 (Colour online) Leaf curling and yellowing symptoms on a Sesbania bispinosa plant in a cotton field, while asymptomatic S. bispinosa plants can be seen in the background.

Materials and methods

Plant sampling and DNA extraction

For the molecular detection of possible virus, symptomatic leaves from S. bispinosa plants showing leaf curling and vein swelling were collected from southern Punjab, Pakistan. Total DNA was extracted from 100 mg of leaf tissues using the CTAB method (Doyle & Doyle, 1987) and resolved on 1% agarose gels prepared in 0.5× TAE buffer. Quantification of DNA was done using a Nanodrop (ThermoFisher Scientific, Waltham, MA).

Amplification, cloning and sequencing of begomovirus and betasatellite

Rolling Circle amplification (RCA) was used to amplify all circular molecules in the DNA extracts of S. bispinosa (Haible et al., 2006). Universal begomovirus primers (Shahid et al., 2007) and universal betasatellite primers (Briddon et al., 2002) were used in PCR reaction to amplify full-length virus genome and betasatellite from the RCA (Rolling circle amplification) enriched product. Upon resolving the PCR product on a 1% agarose gel, the begomovirus primers amplified a product of ~2.8 kb and the betasatellite primers amplified a product of ~1.4kb. The amplified and potentially full-length PCR products were cloned in pTZ57R ⁄T vector (Thermo Fisher Scientific) and were sequenced in entirety using dideoxy nucleotide chain termination sequencing on an Applied Biosystems 3730XL (Thermo Fisher Scientific) DNA sequencer. Clones were completely sequenced, assembled and included for further phylogenetic analysis.

Identification of begomovirus and betasatellite

DNA star (Madison, WI, USA) was used to assemble the cloned sequences. The online database of NCBI was used to retrieve similar sequences and a comparison was done using online nucleotide BLAST search (Altschul et al., 1990). CLUSTAL W (Thompson et al., 1994) of software MEGA 6 (Tamura et al., 2013) was used to perform sequence alignments and to construct neighbour-joining phylogenetic dendrograms with 1000 bootstrap values. Further confirmation was done by performing MUSCLE alignment using sequence demarcation tool (SDT) as a requirement of revised begomovirus taxonomy (Brown et al., 2015).

Results and discussion

Sequence identification, ORF analysis and identification of begomovirus

The RCA enriched products from nucleic acid extracts of symptomatic S. bispinosa plants were confirmed for the presence of begomovirus through PCR amplification using universal begomovirus primers. These primers amplified products of ~2800 bp, which were cloned and fully sequenced. Three independent clones were obtained and their full-length sequences were submitted to NCBI database (www.ncbi.nlm.nih.gov) and are available under accession numbers LN678638, LN713272 and LN713273. These sequences are 2766, 2771 and 2776 nucleotides in length, respectively, and each sequence encodes six open reading frames (ORFs). These six ORFs translate into six distinct proteins above 10 kDa, and their arrangement is similar to the other Old World (OW) monopartite begomoviruses. The coordinates of the ORFs on a circularized viral particle with the predicted sizes of proteins are described in Table 1. The sequences of begomovirus isolated from S. bispinosa were checked for sequence similarity with other available sequences in database using BLASTn (Altschul et al., 1990). The results indicate the sequences are most similar (98.73%) to Pedilanthus leaf curl virus (PeLCV) isolated from a symptomatic plant of soybean in Pakistan (AM948961). The percentage identity was also confirmed using SDT. The present criterion for species demarcation of begomovirus genome is set at nucleotide similarity of 91% (Brown et al., 2015). According to this criterion, the virus that has been found infecting S. bispinosa is an isolate of PeLCV. The isolate descriptors for the virus are Pedilanthus leaf curl virus-Sesbania [Pakistan:Sesbania:2013] which can be abbreviated as PeLCV [PK:Sesbania:13]. A phylogenetic analysis of PeLCV isolates from S. bispinosa was done by aligning them with complete nucleotide sequences of viruses retrieved from the database. The alignment is represented as a neighbour-joining phylogenetic tree in Fig. 2a. The analysis confirms that the isolates of PeLCV are most similar to the isolates from soybean which are also closely related to Radish leaf curl virus (RaLCuV) and Papaya leaf curl virus (PaLCV). The complete phylogenetic analysis, with reliable bootstrap values, indicated that all, new and previously reported, isolates of PeLCV are closely related to a recently identified species of begomovirus, RaLCuV (Tahir et al., 2009; Srivastava et al., 2014).

Table 1. Open reading frames in the Pedilanthus leaf curl virus (PeLCV) and Tobacco leaf curl betasatellite (TbLCB) isolated from Sesbania bispinosa.

ORF	Position (nucleotide coordinates ±5)	Coding capacity (amino acids)	Predicted product size (kDa)
CP	305–1075	256	29.7
V2	97–501	134	15.3
Rep	2609–1524	361	40.6
TrAP	1621–1217	134	15.2
REn	1476–1072	134	15.7
C4	2452–2150	100	10.9
βC1	188–571	127	14.7

Fig. 2 (Colour online) Neighbour joining phylogenetic dendrograms and pairwise percentage identities of Pedilanthus leaf curl virus (PeLCV) and Tobacco leaf curl betasatellite (TbLCB) isolates from Sesbania bispinosa with other similar sequences retrieved from NCBI nucleotide database. The phylogenetic trees are based on pair-wise alignment, done using CLUSTAL W program of MEGA 6 and percentage identities were calculated using sequence demarcation tool (SDT) MUSCLE. The nomenclature of each sequence in the tree is: Name of virus- [Country:Location:Host:Year of sample collection]-Accession number. Both trees are supported by 1000 bootstrap value, represented with each root. a, The phylogenetic tree of whole genome of virus indicates that PeLCV isolated from S. bispinosa is closely related to PeLCV isolated from soybean from Pakistan (green). Overall PeLCV shows similarity with [RaLCuV (red)] and Papaya leaf curl virus [PaLCuV (blue)] isolates from various hosts in Pakistan and India. Tomato mottle virus (ToMoV) is used as an outgroup. b, The graphical representation of pairwise identities based on SDT MUSCLE alignment and the colour table on the right describes the percentage identity that each colour represents. c, The phylogenetic tree of betasatellites shows that TbLCB isolated from S. bispinosa is closely related to the three isolates of TbLCB, one from Pedilanthus and two from tobacco (green). Overall the sequences of TbLCB show association with Radish leaf curl betasatellite (RaLCB) isolates from various hosts in Pakistan and India (red). Cotton leaf curl Multan alphasatellite (CLCuMuA) is used as an outgroup.

Identification and ORF analysis of betasatellite

Mostly begomoviruses belonging to the New World are bipartite as their genome is composed of two components, DNA A and DNA B (Mansoor et al., 2003). However, all efforts to amplify and clone DNA B in the present study were unsuccessful (data not shown). This strongly indicates that the begomovirus infecting S. bispinosa is a monopartite begomovirus and lacks DNA B component. Most of the monopartite begomoviruses in the OW are associated with a symptom/pathogenicity determinant circular ssDNA satellite known as a betasatellite (Mansoor et al., 2003). In the present study, PCR reactions with universal betasatellite primers amplified the expected product of ~1350 bp. The products were cloned and sequenced completely. Five independent clones were obtained and all betasatellite isolates were 1359 bp in length. These sequences were submitted to the database and are available under accession numbers HG983284, HG983283, HG983282, HG983281 and HG965238. Sequence analyses showed that these molecules possessed typical characters of betasatellites. They contain an adenine-rich stretch of nucleotides and a highly conserved region among all betasatellites. These stretches of nucleotides typically range from 738–986 bp and 1243–14 bp respectively, on a circularized satellite genome. The conserved region also contains a nonanucleotide sequence (TAAATT/AC) that marks the origin of replication for these DNA molecules. The pathogenicity determinant protein βC1, with a predicted size of 127 amino acids, is encoded by an ORF, which is present in complementary sense orientation and it is the only ORF present in these betasatellites (Cui et al., 2005; Saeed et al., 2005). A phylogenetic analysis based on the complete nucleotide sequences of the betasatellites obtained here with the selected sequences from database was performed. The analysis showed that the satellites isolated from S. bispinosa are most similar (96% and 96.75%) with two isolates of Tobacco leaf curl betasatellite from pedilanthus and tobacco. Sequences are available in the database under accession numbers AM260465 and AJ316033, respectively (Fig. 2b). Thus, based on this identity, we can conclude that betasatellite associated with S. bispinosa is an isolate of TbLCB and its complete description is TbLCB-[PK: Sesbania:13].

Begomovirus disease complexes are widespread throughout the OW and are increasingly found in new locations and hosts. The data presented here shows that the leaf curl disease of S. bispinosa is associated with a monopartite begomovirus PeLCV and TbLCB. PeLCV has previously been found to be associated with soybean (Ilyas et al., 2010). PeLCV and TbLCB disease complexes have also been reported from an ornamental shrub Pedilanthus tithymaloides in Pakistan (Tahir et al., 2009). To the best of our knowledge, this report from Pakistan is the first to describe the occurrence of PeLCV and TbLCV on S. bispinosa worldwide.

Acknowledgement

This research was funded in part through the Pakistan–U.S. Cotton Productivity Enhancement Program, USDA Agricultural Research Service Project No. 58-6402-0-178F. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.