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. 2020 May 6;10(5):235. doi: 10.1007/s13205-020-02220-6

Complete genome sequence of a new bipartite begomovirus associated with leaf curl disease of Capsicum annum

Anurag Kumar Sahu 1, Neeti Sanan-Mishra 1,✉
PMCID: PMC7203235  PMID: 32399385

Abstract

The complete bipartite genome, consisting of DNA-A and DNA-B, of a novel begomovirus isolate associated with apical leaf curling and crinkled leaf disease of Chili (Capsicum annum) from New Delhi, India was cloned and sequenced. The sequence of DNA-A (2737 nt) and DNA-B (2692 nt) of the virus was submitted to NCBI, USA under the accessions MK069591 & MG597211, respectively. Sequence identity of the common region (CR) and presence of identical iterons (GAGTG) between the DNA-A and DNA-B clones indicate that they constitute a related pair. The virus corresponds to a novel species of tomato leaf curl virus and sequence analysis has ruled out the involvement of recombination events in its evolution. Therefore, we report the complete nucleotide sequence of a new bipartite begomovirus infecting Capsicum annum, a vegetable crop communally cultivated throughout India.

Electronic supplementary material

The online version of this article (10.1007/s13205-020-02220-6) contains supplementary material, which is available to authorized users.

Keywords: Capsicum annum, Bipartite begomovirus, Genome sequencing, Phylogeny

Geminivirus is the largest family of plant viruses with circular, single-stranded DNA (ssDNA) genomes packaged within geminate particles (Hanley-Bowdoin et al. 2000). They are categorized into nine genera based on host range, vector transmission and genome organization: Becurtovirus, Begomovirus, Capulavirus, Curtovirus, Eragrovirus, Grablovirus, Mastrevirus, Topocuvirus and Turncurtovirus (Varsani et al. 2017). Members of the genus Begomovirus are transmitted by whiteflies (Bemisia tabaci) and they mostly prevail in the tropical and sub-tropical regions of the world (Hanley-Bowdoin et al. 1999; Markham et al. 1994).

Begomoviruses have either monopartite (a single DNA-A-like component) or bipartite (two DNA components: DNA-A and DNA-B) genomes (Zerbini et al. 2017). Most of the monopartite begomoviruses are associated with single-stranded satellite DNA viz. alpha- and beta-satellites (Mansoor et al. 2006; Kumar et al. 2015). Both types of satellite molecules depend upon their helper virus for replication and/or movement (Muthupandi et al. 2019; Kumar et al. 2017). The bipartite virus genes are resident on two different circular ssDNA molecules (DNA-A, DNA-B) each of about 2.5–2.7 kb (Brown et al. 2012). DNA-A-encoding proteins are responsible for replication, encapsidation and vector transmission and DNA-B-encoding proteins have movement-related functions (Shahid et al. 2019). The two DNA components do not have sequence identity to each other, except for a common region (CR), which is highly conserved (generally above 90% sequence identity) between the two components of the same species. The CR includes recognition sequence for viral replication-associated protein (Rep) as well as regions responsible for transcription of viral genes (Hanley-Bowdoin et al. 2000).

Chili leaf curl disease (ChiLCD) caused by infection of begomoviruses is a major concern as chili (Capscicum annuum L.) is the most widely used spice of the world and an important cash crop of Solanaceae family that is cultivated extensively in India. Infection with tomato leaf curl virus (ToLCV) causes 90–100% yield loss in chili (Chavan et al. 2015). In this study, we report the identification of a novel bipartite begomovirus associated with ChiLCD. The methodology used and results are presented briefly.

Twenty samples with typical ChiLCD symptoms were collected from different places in New Delhi, India in the year 2017. To investigate the type of virus in the infected samples, total DNA was extracted from the infected tissues using the cetyl trimethyl ammonium bromide (CTAB) method (Manen et al. 2005) and subjected to PCR using a pair of degenerate primers specific to the coat protein region (AC-1048F 5ʹGCCYATRTAYAGRAAGCCMAG3ʹ and AV-494R 5ʹGCCYATRTAYAGRAAGCCMAG3ʹ) of begomovirus. Amplification of DNA fragment confirmed the begomovirus infection (data not shown). The presence of betasatellite associated with the virus was also checked in the same samples using specific PCR primers Beta01/Beta02 (Briddon et al. 2002), but no amplification indicated that betasatellite molecules were not present.

To isolate and characterize the viral genome, rolling circle amplification (RCA) was carried out using TempliPhi DNA amplification kit (GE Healthcare). The RCA products were digested with restriction endonucleases, viz. EcoRI, BamHI, KpnI, HindIII or SalI, respectively. Fragments of ~ 2.7 kb were obtained with EcoRI and cloned into the pTZ57R/T vector (Fermentas) linearized with the same restriction enzyme (Suppl. Fig. S1). Five positive colonies were selected for sequencing. The DNA-A and DNA-B genomes of 2737 nt and 2692 nt, respectively, were identified in the same sample. All the sequences showed 98–99% identity to each other, therefore, only one sequence each, of DNA-A and DNA-B was used for further analysis. The DNA-A contained six predicted open reading frames (ORFs AV1, AV2, AC1, AC2, AC3, and AC4), while DNA-B contained two predicted open reading frames (ORFs BV1, and BC1).

The cloned viral sequences were subjected to NCBI BLASTn algorithm using default parameters and begomovirus sequences showing best match were selected. Genomic sequences were aligned with MUSCLE (Brown et al. 2015) implemented in MEGA v.7.0 (Kumar et al. 2016). Maximum-likelihood (ML) phylogenetic trees for each data set were generated using PAUP* 4.0 (Swofford and Sullivan 2003) with tree-bisection-recombination branch-swapping in each case. The best-fit model of nucleotide substitution for each data set was determined using MODELTEST (Posada and Crandall 1998). To assess the support for individual nodes on the phylogenies, a bootstrap analysis was performed utilizing 1000 replicates under the ML substitution model in each case. To determine percentage pairwise identity between the cloned sequence and the selected begomovirus sequences, CLUSTAL W method in SDT v.1.0 (Muhire et al. 2014) was used. Phylogenetic trees based on DNA-A and DNA-B alignments were generated by Bayesian inference using MrBayes v.3.0b4 (Ronquist and Huelsenbeck 2003) with the HKY + G nucleotide substitution and MrModel test v.2.2 (Nylander 2004) using TN-93 + G + I nucleotide substitution model, respectively.

The full-length sequence of cloned DNA-A showed maximum 91% sequence identity with the Tomato leaf curl Hsinchu virus (DQ866131) and Tobacco leaf curl Pusa virus (KM383734) (Fig. 1a), while it showed 78–88% nucleotide sequence identity with other reported begomoviruses (KX885030, JX460805, LC051120, KM383734, HM007104, KM383745, and DQ866131) (Fig. 1b). Based on the threshold level of 91% nucleotide sequence similarity, established by the Geminiviridae study group of the ICTV (Brown et al. 2015), the virus isolate identified in this study was categorized as a novel begomovirus species. Nucleotide sequence comparison of DNA-B sequences with other begomoviruses showed 87–98% nt sequence identity to other ToLCV isolates (Fig. 2a). ML phylogenetic analysis of complete DNA-B sequence demonstrated that its clusters with Tomato leaf curl New Delhi virus (HM989846) from India (Fig. 2). Sequence identity of the common region (CR) as well as the presence of identical iterons (GAGTG) between the DNA-A and DNA-B clones indicates that they are related pair. Thus, the identified DNA-A corresponds to the identified DNA-B indicating that they constitute a bipartite virus. Their individual sequences have been deposited in GenBank under the accession numbers MK069591 (DNA-A) and MG597211 (DNA-B), respectively.

Fig. 1.

Fig. 1

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a ML phylogenetic tree showing the relationship between DNA-A (MK069591) of the novel begomovirus and the selected GeneBank begomovirus sequences. The tree was constructed using MEGA v.7.0 with bootstrap value of 1000 replicates. Horizontal lines represent the evolutionary distance between the sequences measured in terms of nucleotide substitutions. b Graphical representations of percentage pairwise genome scores and nucleotide identity of cloned DNA-A with the most closely related begomoviruses using SDTv1.2 program. The DNA-A sequence (MK069591) of the novel begomovirus identified in this study is indicated by arrow

Fig. 2.

Fig. 2

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a ML phylogenetic tree showing the relationship between DNA-B sequence (MG597211) of the novel begomovirus and the GeneBank begomovirus sequences showing best match to it. The tree was constructed using MEGA v.7.0 with bootstrap value of 1000 replicates. Horizontal lines represent the evolutionary distance between the sequences measured in terms of nucleotide substitutions. b Graphical representations of percentage pairwise genome scores and nucleotide identity of cloned DNA-B with the most closely related begomoviruses using SDTv1.2 program. The DNA-B sequence (MG597211) of the novel begomovirus identified in this study is indicated by arrow

Emergence and evolution of new begomoviruses involves mutation, pseudorecombination and recombination (Silva et al. 2014). Recombination is known to play a major role by increasing genetic variation (George et al. 2015; Sahu et al. 2018) thus it is important to perform recombination studies when analyzing new begomoviruses. To evaluate whether the identified DNA-A and DNA-B sequences show evidence of recombination, the sequences were analyzed using the Recombination detection program (RDP v4) (Martin et al. 2010). Full-length sequences of 26 begomovirus (in the database) showing best match to the cloned sequences were used in a pairwise scanning approach (https://darwin.uvigo.es/rdp/rdp.html) employing RDP, GENECONV, MaxChi, Chimaera, Bootscan and 3Seq with the Bonferroni corrected P value cut-off of 0.01. The default detection thresholds were employed in all cases. The results revealed no evidence of significant recombination events (Suppl. Table S1).

We, therefore, report a new member of the bipartite begomovirus species naturally infecting Capsicum annum (chili). There is need for a more comprehensive study to identify the begomovirus diversity in the country and to evaluate the contribution each makes to crop losses. This will enable studies on understanding virus evolution in the perspective of host diversification and help in developing control strategies.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary file1 (DOC 60 kb) (60KB, doc)
13205_2020_2220_MOESM2_ESM.tif (299.8KB, tif)

Supplementary Fig. S1. Rolling circle amplification (RCA) of virus infected chili samples and cloning of the amplified fragment in pTZ57R/T vector. A. Rolling circle amplification (RCA) of six individual virus infected chili samples. The amplification product of ~2.7 Kb is shown. B. The RCA products were cloned and checked by restriction digestion. The enzymes used for digestion are mentioned over each lane. lane 1: uncut (UC) plasmid; lane 2: pTZ57R/T-DNA-A digested with EcoRI. Two bands correspond to the plasmid backbone and the 2.7 Kb insert; lane 3: pTZ57R/T-DNA-A digested with HindIII. Single band correspond to the linearized plasmid backbone containing the insert; lane 4: 1 Kb marker (MW) (TIF 299 kb)

Acknowledgements

AKS is thankful for financial support from the DBT-RA Program in Biotechnology and Life Sciences.

Funding

This study was funded by DBT-RA 2016, Department of Biotechnology, Ministry of Science and Technology, Govt. India.

Compliance with ethical standards

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Contributor Information

Anurag Kumar Sahu, Email: anuragsahhu@gmail.com.

Neeti Sanan-Mishra, Email: neeti@icgeb.res.in.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary file1 (DOC 60 kb) (60KB, doc)
13205_2020_2220_MOESM2_ESM.tif (299.8KB, tif)

Supplementary Fig. S1. Rolling circle amplification (RCA) of virus infected chili samples and cloning of the amplified fragment in pTZ57R/T vector. A. Rolling circle amplification (RCA) of six individual virus infected chili samples. The amplification product of ~2.7 Kb is shown. B. The RCA products were cloned and checked by restriction digestion. The enzymes used for digestion are mentioned over each lane. lane 1: uncut (UC) plasmid; lane 2: pTZ57R/T-DNA-A digested with EcoRI. Two bands correspond to the plasmid backbone and the 2.7 Kb insert; lane 3: pTZ57R/T-DNA-A digested with HindIII. Single band correspond to the linearized plasmid backbone containing the insert; lane 4: 1 Kb marker (MW) (TIF 299 kb)

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