Citharexylum spinosum L. (family: Verbenaceae), commonly known as fiddlewood, is a large shrub or small tree with fragrant flowers native to the Caribbean region [1]. The species was introduced to and got naturalized in India where it is generally grown in gardens and occasionally found in wild [1]. The tree is useful in treating various ailments due to its medicinal properties and its wood is used for making stringed musical instruments [2].

Begomoviruses (family: Geminiviridae) are single-stranded DNA genome-containing viruses that cause economically important diseases in a wide range of crops worldwide. Broadly, they are categorized as Old World and New World begomoviruses based on their genome organization and geographic distribution. Predominantly, the Old World begomoviruses contain monopartite genome and the New World begomoviruses contain bipartite genome with DNA-A and DNA-B components. Whiteflies efficiently transmit begomoviruses and infected plants typically depict symptoms, like leaf yellowing, leaf curling, and stunted growth [3].

During March 2023, leaf curl disease was observed in C. spinosum plants growing in the Telangana State Forest Academy campus, Hyderabad, Telangana, India. The diseased plants exhibited severe leaf curling and leaf mottling symptoms (Fig. 1) and the disease incidence was 91.8% (45 diseased of 49 plants observed). Symptomatic leaf samples were collected from three individuals and total DNA was isolated from 100 mg of collected samples using cetyl trimethylammonium bromide (CTAB) protocol [4]. PCR amplification of isolated DNA using begomovirus coat protein (CP) gene-specific degenerate primers [5] yielded positive bands. However, positive amplification could not be obtained for alpha- [6] and betasatellite-specific universal primers [7]. Further, isolated DNA from one of the samples was subjected to rolling circle amplification (RCA) using phi29 DNA polymerase (Thermo Fisher Scientific). Amplicons obtained after RCA were digested using the restriction endonuclease BamHI to obtain the putative monomers of begomovirus genome (ca. 2.8 kb), which were ligated to BamHI-digested pUC18 vector and transformed into competent cells of Escherichia coli strain DH5α. Plasmids were isolated from positive colonies and the presence of desired insert was confirmed after restriction digestion with BamHI and BglI. Isolated plasmids from one of the positive colonies with the desired insert was outsourced for sequencing of insert through primer walking strategy (University of Delhi South Campus, New Delhi). The obtained genome sequence along with a few selected begomoviral sequences, including those of closely related viruses identified in BLASTn search, retrieved from National Center for Biotechnology Information (NCBI) were aligned using CLUSTALW tool and subjected to maximum-likelihood (ML) phylogenetic tree construction using the best-fit TN93 + G model with 500 bootstrap replicates in MEGA 7 (v 7.0.26) [8]. Sequence identity matrix was obtained for the MUSCLE aligned sequences using Species Demarcation Tool (v 1.2) [9]. For detection of possible recombination events, the obtained genome sequence along with the top 100 hits resulting from BLASTn analysis of obtained genome were aligned and imported into Recombination Detection Program (RDP) (v 4.101) [10]. Only recombination events detected by more than five methods were considered reliable.

Fig. 1
figure 1

Symptoms of leaf curl disease in Citharexylum spinosum growing in Telangana, India. A A diseased tree showing severe leaf curl symptom. Close-up view of twigs showing B leaf curling and C. mottling symptoms

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The obtained genome sequence was 2767 nucleotide (nt) long (GenBank accession number: OR437368) and contained two open reading frames (ORFs) (V1 and V2) in the viral strand and four ORFs in the complementary strand (C1–C4), resembling the typical genome organization of Old World begomoviruses [3]. Besides, two small non-canonical ORFs (C5 and C6), similar to the ones detected in a few begomoviruses [11,12,13], were determined in the viral complementary strand. The 302 nt-long intergenic region (IR) contained two copies of the replication-associated protein (Rep)-binding iteron sequence “GGWCYC” (the corresponding iteron-associated domain in Rep is MPPKRFLIN), identical to tomato leaf curl Kerala virus (ToLCKeV) [14]. Following the iteron are the ‘TATA’ box and an inverted repeat sequence capable of forming the stem loop structure separated by the conserved nonanucleotide sequence (TAATATTAC). The length of each ORF and predicted motifs in and molecular weight of encoded proteins are provided in Supplementary Table 1. Coat protein sequence-based identity matrix revealed that the coat protein encoded by the obtained genome sequence shared a maximum of 97.7% identity with the corresponding sequence of Duranta leaf curl virus (QJX74439.1) (Supplementary Fig. S1). Genome sequence-based identity matrix revealed the maximum sequence identity (89.7%) of obtained genome sequence with that of a papaya leaf crumple virus (PaLCrV) isolate (KR071789.1) (Fig. 2a; Supplementary Table 2). Based on the < 91% nt sequence identity criterion (of the complete genome sequence in case of monopartite begomoviruses) [15], the identified virus from C. spinosum can be regarded as a novel begomoviral species for which the name “Citharexylum leaf curl virus” (CitLCuV) is proposed. Phylogenetic tree placed CitLCuV in a sister clade to PaLCrV isolates and these together with rose leaf curl virus isolates formed a distinct subclade (Fig. 2b). Recombination analysis identified a putative recombination event in CitLCuV genome at nt position 70–2106, with tomato leaf curl Kerala virus (KF551575.1) as major parent and Chenopodium leaf distortion virus (MN423112.1) as minor parent (Supplementary Fig. S2; Supplementary Table 3).

Fig. 2
figure 2

A Identity matrix showing the sequence similarities of Citharexylum leaf curl virus (CitLCuV) with its closely related begomoviruses. B Maximum-likelihood tree showing the phylogenetic relationship of CitLCuV with its closely related begomoviruses. The tree was constructed using the best-fit TN93 + G model with 500 bootstrap replicates. Only bootstrap values more than 50% are indicated. DNA-A sequence of cotton leaf crumple virus (CLCrV), a bipartite begomovirus, was used as outgroup during tree construction. Expansions of virus names are provided in Supplementary Table 4. CitLCuV identified in this study is shown in bold

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In conclusion, CitLCuV is a novel monopartite Old World begomovirus associated with leaf curl disease of C. spinosum, an exotic species to India. Though satellites could not be detected in symptomatic samples through PCR assays, further validation is required to ascertain the same through Next-Generation Sequencing. Further studies are needed to understand the biological properties and geographical distribution of CitLCuV.