Eustoma russellianum commonly known as lisianthus, is an herbaceous flowering plant that belongs to the Gentianaceae family (Wazir et al. 2014). The popularity of lisianthus is increasing in India, and the area under cultivation is gradually expanding (Bhatia and Sindhu 2019; Lakshmaiah et al. 2019; Rehana and Bala 2022). Lisianthus cultivation faces challenges from various fungal (Bhatia et al. 2020; Wu et al. 2023; Hanagasaki et al. 2023) and viral diseases (Chen et al. 2000; Jan et al. 2003; Beikzadeh et al. 2011; Cohen et al. 2001; Taniguchi et al. 2023) worldwide leading to significant economic losses for growers. There are no reports of viral diseases affecting lisianthus in India. In this study we report the identification and characterization of a begomovirus affecting lisianthus in India.

Seven leaf samples from lisianthus plants exhibiting symptoms typical to begomovirus infection such as vein thickening, leaf curling, and reduction in leaf size, yellow mosaic and stunted growth were collected (designated as LIS-1 to 7 isolates) along with an asymptomatic leaf sample from Vensai Floritech (13°29’00.4"N 77°28’18.5"E) (Bengaluru, India) (Fig. 1). To confirm the causal agent, total genomic DNA was isolated from the lisianthus samples (Lodhi et al. 1994) and subjected to PCR amplification using degenerate primers specific to DNA-A (PAR1c496/PAL1v1978) and DNA-B (PBL1v2040/PCRc1) as described by Rojas et al. (1993). The amplification yielded PCR amplicon of ~ 1.2 kb, specific to begomoviruses with respect to DNA-A. However, there was no amplification with the DNA-B specific primers, indicating the virus associated with symptomatic lisianthus plants is a monopartite begomovirus. All the samples were also subjected to PCR for detecting the presence of satellite molecules i.e., betasatellite (Briddon et al. 2002) and alphasatellite (Bull et al. 2003) using specific primers but unsuccessful for amplification of both satellite molecules.

Fig. 1
figure 1

Lisianthus infected plants showing begomoviral symptoms; (A) Reduction in leaf size, (B) Leaf curling as compare to (C) healthy under field conditions

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Further, one representative sample (LIS-1 isolate) was used for complete genome amplification by rolling circle DNA amplification (RCA) (Inoue-Nagata et al. 2004). The amplified RCA product (2 µL) was digested with HindIII restriction enzyme to obtain a monomeric unit of ~ 2.7 kb. The digested product was cloned into the linearized pUC19 plasmid with the respective sites. The recombinant clones were confirmed by HindIII restriction digestion and sequenced by the primer walking method at Medauxin Pvt. Ltd., Bengaluru, Karnataka, India. The obtained complete genome sequence was assembled using different bioinformatic tools (BioEdit, ClustalX2, and SeaView), and sequence similarities were checked at the NCBI database using BLASTn (http://www.ncbi.nlm.nih.gov).

The complete DNA-A genome sequence of LIS-1 isolate was deposited in NCBI GenBank under the accession number OR371601 and its genome consists of 2754 nt with seven open reading frames (ORFs), which were identified by using the ORF finder tool (http://www.Ncbi.nlm.nih.Gov/gorf/gorf.html). Analysis showed that viral genome (DNA-A) codes for seven potential ORFs, two [V2 and V1 (CP)] on virion sense strand and five [C1 (Rep), C2 (TrAP), C3 (REn), C4 and C5] on complementary sense strand. These two strands are separated by intergenic region (IR), which harbored a predicted stem-loop structure containing nonanucleotide sequence TAATATTAC which constitutes the origin of viral DNA replication (Supplementary Tables 1 and Supplementary Fig. 1). Pairwise nt and amino acid (aa) identities for the LIS-1 isolate from lisianthus were compared with selected begomoviruses sequences retrieved from GenBank using the Sequence Demarcation Tool (SDT) (SDT version 1.2) (http://web.cbio.uct.ac.za/brejnev/) (Muhire et al. 2014). Sequence analysis revealed that the LIS-1 isolate exhibited nt sequence identity of 87.8–91.0 per cent for the complete genome with sida yellow vein Madurai virus (SIYVMV). The highest nt identity (91.0%) indicates that the LIS-1 isolate represents a strain of SIYVMV infecting lisianthus (Brown et al. 2015; Table 1 and Suppl. Table 2). This is further supported by SDT analysis (Fig. 2b).

Table 1 Pairwise nucleotide and amino acid identities between the begomovirus isolated from lishanthus (SIYVMV-[IN:Kar:Dod:Lis:23]) and selected begomoviruses
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Fig. 2
figure 2

(A) Phylogenic tree based on the complete nucleotide sequence of SIYVMV isolate from lisianthus (IN:Kar:Dod:Lis:23;OR371601) and other selected begomoviruses. (B) Nucleotide sequence identity matrix obtained from the same dataset using Sequence Demarcation Tool. and (C) recombination breakpoint analysis of SIYVMV (OR371601) isolate with other selected begomoviruses isolates reported across the world

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Based on the ICTV guidelines for begomovirus nomenclature (Fauquet and Stanley 2005), we propose the name “Sida yellow vein Madurai virus-[India:Karnataka:Doddaballapura:Lisianthus:2023]”, abbreviated as SIYVMV-[IN:Kar:Dod:Lis:23], for the isolate.

Amino acid identities of the proteins encoded by the isolate with those encoded by other begomoviruses are listed in Table 1.

A phylogenetic tree generated using the Neighbor-Joining method by MEGA11 (Tamura et al. 2021) revealed that SIYVMV-[IN:Kar:Dod:Lis:23] (OR371601) clustered with sida yellow vein Madurai virus (SIYVMV) infecting sida plants reported from Tamil Nadu, India (Fig. 2a). Recombination analysis of SIYVMV-[IN:Kar:Dod:Lis:23] using RDP 5 (Martin et al. 2021) revealed a single significant intra-specific recombination event occurring at the 14th to 2087th nt position in the DNA-A genome of SIYVMV-[IN:Kar:Dod:Lis:23]. This recombinant fragment descends from okra enation leaf curl virus (OELCuV: MK069435) and sida yellow vein Madurai virus (SIYVMV: OM141480) as the major and minor parents, respectively (Table 2; Fig. 2c). This report provides evidence of the association of recombinant begomovirus with lisianthus for the first time in India. Furthermore, symptomatology, characterization, phylogeny and recombination analysis indicated that leaf curl disease of lisianthus is caused by a strain of SIYVMV.

Table 2 Recombination analysis of the begomovirus isolate from lisianthus (SIYVMV-[IN:Kar:Dod:Lis:23]).
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