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. 2020 Mar 16;10(4):169. doi: 10.1007/s13205-020-2159-9

Identification of Chilli leaf curl virus and associated betasatellite infecting Osteospermum fruticosum in Rajasthan, India

Megha Mishra 1, Rakesh Kumar Verma 1, R K Gaur 2,✉
PMCID: PMC7076104  PMID: 32206503

Abstract

A monopartite begomovirus associated with betasatellite was identified from Osteospermum fruticosum (Cape Daisy) showing severe yellowing vein net symptoms in Rajasthan, India through molecular characterization. The DNA-A shared the highest nucleotide (96.61%) identity to Chilli leaf curl Ahmedabad virus (KM880103), while the betasatellite depicted the highest sequence similarity (99.28%) to Chilli leaf curl betasatellite (JF706231, 99.28%). Based on the sequence identity with other begomoviruses known to date, they were recognized as Chilli leaf curl virus (CDI, MH355641) and Chilli leaf curl betasatellite (CDB1, MH355642), respectively. Phylogenetic analysis showed that DNA-A (CD1) clustered with ChiLCV Goa (KP235539), whereas the betasatellite (CDB1) clustered with ChiLCB Jodhapur (JF70623). Recombination events were observed among the clades of ChiLCV, showing intragenic recombination in Rep (C1) and coat protein (V1/AV1) regions. To our knowledge, this is the first report of ChiLC begomovirus strain affecting O. fruticosum.

Keywords: Cape daisy, Begomovirus, Chilli leaf curl virus, Betasatellite, Recombination

The family Geminiviridae is classified into nine genera, Becurtovirus, Begomovirus, Capulavirus, Curtovirus, Eragrovirus, Grablovirus, Mastrevirus, Topocuvirus and Turncurtovirus (Zerbini et al. 2017) based on vector transmission, host range and genome organization (Khan et al. 2013). Begomovirus is the largest genus of the family Geminiviridae, and it causes severe diseases in many of the economically important crops, weeds and ornamental plants throughout the world (Inoue-Nagata et al. 2016). Begomoviruses are circular single-stranded DNA (ssDNA) viruses that are encapsidated in icosahedral geminate particles and transmitted by whitefly (Bemisia tabaci). The viral genome is characterized as monopartite (DNA-A) or bipartite (DNA-A and DNA-B) components, each component ranges from 2.7 to 3.0 Kb. These component share a characteristic intergenic region termed as common region (CR) of ~ 200 nt in length and contain a nanonucleotide stem-loop structure sequence ‘TAATATTAC’ (Zhou 2013; Zerbini et al. 2017). The majority of bipartite begomoviruses belongs to the New World (NW), while the monopartite viruses belong to the Old World (OW) (Shahid et al. 2019). DNA-A encodes six genes, coat protein (CP) and V2/AV2 on the virion-sense strand, while the complementary strand encodes the replication associated protein (Rep/C1), transcriptional activator protein (TrAP/C2), replication enhancer protein (Ren/C3) and C4 protein. In DNA-B, the complementary strand encodes the movement protein (MP) and nuclear shuttle protein (NSP) on the virion-sense strand. Both the proteins are involved in cell to cell movement of the virus and the transport of newly synthesized viral DNA from the nucleus to the cytoplasm (Shahid et al. 2019). Monopartite begomoviruses are frequently associated with novel DNA components, namely betasatellite and alphasatellite. These satellite DNAs are approximately half the size of the begomovirus DNA-A. Betasatellite (~ 1350 nts) encodes a single gene, βC1, which plays crucial roles in suppression of host gene silencing and symptom induction (Briddon and Stanley 2006; Zhou 2013). Alphasatellites (~ 1400 nts) encode alpha-rep and replicate independently but have no known role in pathogenesis (Romay et al. 2010; Shahid et al. 2016).

During a survey in 2017–2018 for begomovirus infection, severe yellow vein net and mild downward curling of Cape Daisy (O. fruticosum) were observed in the Shekhawati region of Rajasthan, India (Fig. 1). To confirm the presence of begomovirus infection, total DNA was extracted from 20 infected leaf samples and a healthy leaf by using the CTAB method (Doyle and Doyle 1987). Qualitative and quantitative estimations of the extracted DNA were performed using agarose gel electrophoresis and NanoDrop (Thermo Scientific, US), respectively. The existence of begomovirus were assessed by PCR using the degenerate primer DNA101/DNA102 (Bull et al. 2003). Among the collected 20 samples, only 9 samples were found to be positive producing an amplicon of ca. 650 bp. In contrast, amplifications from the non-symptomatic plants were uniformly negative. PCR was also used to amplify the betasatellite using universal primer pair Beta01/Beta02 (Briddon et al. 2002) was used. All attempts for the detection of DNA-B and alphasatellite using degenerate primer pairs PCRc1/PBL1v2040 (Cui et al. 2004) and UN101/UN102 (Wu and Zhou 2005), respectively, were unsuccessful. The full-length genomic DNA component of the positive samples was further amplified using the TempliPhi kit (GE Healthcare) as per the manufacturer’s instructions. The positive rolling circular amplification (RCA) products were digested with HindIII and PstI endonucleases, cloned into pUC19 vector and sequenced (Xcelris Genomics, Ahmedabad, India). The sequences were analysed using nucleotide–nucleotide comparison of BLAST program (https://blast.ncbi.nlm.nih.gov). Furthermore, the sequences were examined for the begomovirus specific Open reading frames (ORFs) using NCBI ORF finder and conserved nonanucleotide sequence, (https://ncbi.nlm.nih.gov/orffinder). A total of 37 full-length sequences of ChiLCV and 18 sequences of ChiLCB originating from India were retrieved from NCBI GenBank database for the similarity identity study. Furthermore, these sequences were analysed using MEGA 7.0 software (Kumar et al. 2016) to determine the phylogenetic interference. Recombination analysis was performed using seven automated algorithms, namely RDP, GENECONV, BOOTSCAN, MAXCHI, CHIMAERA, SISCAN and 3SEQ, which are available in the Recombination Detection Program 4.2 version (Martin et al. 2015; Nehra et al. 2019). Recombination breakpoints supported by at least three or more algorithms with the highest accepted p value of 0.05 were considered as positive.

Fig. 1.

Fig. 1

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Typical Chilli leaf curl virus symptoms on Cape Daisy. a Cape Daisy healthy plant. b Cape Daisy plant showing infection

The present study provides the recombination breakpoints, phylogeny and the complexity of ChiLCV associated with the vein net disease of Cape Daisy. The sequences of 2.7 kb and 1.3 kb clones were submitted to GenBank database and assigned the accession numbers MH355641 (isolate CD1) and MH355642 (isolate CDB1). The 2.7 kb clone (MH355641) exhibited the typical arrangement of DNA-A of begomovirus encompassing six ORFs (AV1, AV2, AC1, AC2, AC3, AC4) and was accompanied by a conserved nonanucleotide sequence TAATATTAC. The 1.3 kb clone (MH355642) resembled the begomovirus associated betasatellite. BLAST alignment analysis revealed that the genomic DNA-A component (MH355641) has the high level of nucleotide similarity with Chilli leaf curl virus (96.61%; KM880103), whereas the betasatellite component (MH355642) has the maximum sequence identity with Chilli leaf curl betasatellite (99.28%; JF706231). The maximum likelihood (ML) phylogenetic relationship of the CDI (MH35564) isolates showed a cluster with the ChiLCV isolate Goa (KP235539) (Figs. 2a, 3a). However, the CDB1 (MH355642) of Cape Daisy was found to cluster with ChiLCBJodhpur (JF706231) (Figs. 2b, 3b). Three recombination breakpoints were detected in the DNA-A component of ChiLCV isolate CD1, but none were observed in the ChiLCB isolate CDB1 (Table 1) Our analysis indicated that the majority of the recombination breakpoints are in the C1 and V1/AV1 regions of DNA-A genomic component, which provides the signal of interspecific recombination in the evolution of begomovirus (Kumar et al. 2015; Nehra et al. 2019).

Fig. 2.

Fig. 2

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The maximum likelihood (ML) phylogenetic tree of aligned complete nucleotide sequences showing relationship among ChiLCV and betasatellite isolates from India, The scale bar represents the number of differences between sequences. a DNA-A. b Betasatellite

Fig. 3.

Fig. 3

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Colour pairwise nucleotide identity matrix between the sequences used in this study. a DNA-A. b Betasatellite

Table 1.

Summary of recombination breakpoints in begomovirus DNA-A isolates, calculated by different algorithms implemented in RDP v 4.0 with optimum p value

Breakpoints Recombinant sequence(s) Minor parental sequence(s) Major parental sequence(s) Recombination detection method p value
2703-536 MH355641:Sikar-Cape_DAISY Unknown MF737343: sikar-Chilli RGBMCS3SEQ 8.36E−28
1741-1815 MH355641:Sikar-Cape_Daisy Unknown MF737343: Sikar-Chilli RGBMC3SEQ 1.50E−08
1215-1740 MH355641:Sikar-Cape_Daisy HM140364: New Delhi-papaya MH346125: Varanasi-chilli RMCS3SEQ 5.18E−20
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The occurrence of ChiLCV and their respective betasatellites in Cape Daisy may cause a serious threat to many economically important crops. This concern may be due to the increased population of whiteflies and the high recombination events of the DNA-A component of begomoviruses (Thakur et al. 2018). The enhanced agro-climatic fluctuations in India may be responsible for the extensive population of the whitefly and the substantial growth of begomoviruses (Malathi et al. 2017; Thakur et al. 2018). ChiLCV is one of the most predominant begomoviruses, and it has a widespread host range in association with the betasatellite (Malathi et al. 2017). So far, no report is available to establish the infection of ChiLCV in the Cape Daisy plant. Our understanding about diversity, genomic architecture, phylogeny and the recombination events in ChiLCV DNA-A as well as the associated satellites may provide new insights for the development of appropriate virus management strategies.

Contributor Information

Megha Mishra, Email: meghamishra1228@gmail.com.

Rakesh Kumar Verma, Email: rkwat4@yahoo.com.

R. K. Gaur, Email: gaurrajarshi@hotmail.com

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