Introduction

Withania somnifera (Syn: ashwagandha, suranjan, punir, winter cherry, Indian ginseng) is an evergreen, xerophytic, woody, short, perineal shrub that belongs to the family Solanaceae. It is naturally found in drier regions ranging from the Mediterranean through tropical Africa through South Africa and from the Canary and Cape Verde Islands to the Middle East and Arabia, Afghanistan, Baluchistan, Pakistan, Sri Lanka, China, Nepal and India. In India, it is extensively distributed in Uttar Pradesh, Punjab, Maharashtra, West Bengal, Gujarat and Rajasthan (Kulkarni and Dhir 2008). This plant is a rich source of some important secondary metabolites like, steroids, alkaloids, flavonoids, phenolics, saponins, and glycosides, etc., which are widely used in traditional medicine (Saleem et al. 2020). It has broad array of pharmacological properties, such as an immunomodulatory, anti-oxidant, anti-inflammatory, antimicrobial, antistress, antitumor, neuroprotective, cardioprotective, antihypertensive, and antidiabetic activities (John 2014). In the last 2 decades, an active component of Withaferin A (WFA) has shown tremendous cytotoxic activity suggesting its potential as an anti-carcinogenic agent in the treatment of several cancers and Alzheimer’s disease (Dutta et al. 2019). The chemical profile and pharmacological activities of W. somnifera have been extensively reported. Several clinical studies have reported its safety for use in humans (Saggam et al. 2021). Its herbal formulations are available in the market, named as, ashwagandharishta, and stresswin.

Begomovirus is a genus of plant viruses belongs to the family Geminiviridae. It has exhibited its potential prevalence from ornamental, vegetable, agricultural to medicinal and aromatic plants (MAPs) (Brown et al. 2015). The transmission of begomovirus is carried out generally by whiteflies (Basak 2016). Its genome consists of closed, single-stranded, circular DNA. On the basis of presence of one or two genomic components, they are divided into monopartite (DNA-A) or bipartite (DNA-A and DNA-B) begomoviruses (Fauquet et al. 2008). DNA-A virion-sense strand encodes two open reading frames (ORFs) (AV1 and AV2) and four in the complementary sense (AC1, AC2, AC3 and AC4) and five ORFs (AC1, AC2, AC3, AC4 and AC5) in New World and Old World begomoviruses, respectively. DNA-B encodes two ORFs, BV1 and BC1 in virion-sense and complementary-sense strands, respectively. Both the genomic components (DNA-A and DNAB) share 200 nt common region (CR), which is present within the intergenic region, where replication origin is present. Begomoviruses are often found to be associated with satellite DNA molecules such as betasatellites, alphasatellites and/or deltasatellites (Lozano et al. 2016). Recent reports have shown the growing incidences and threatening effect of begomoviruses infection on medicinal and aromatic plants (Saeed and Samad 2017).

Withania somnifera has been often reported to be infected by fungal diseases such as Alternaria dianthicola, Psuedocercospora fuligena, Choanephora cucurbitarum, etc. (Maiti et al. 2007; Saroj et al. 2012, 2014). The carmine red spider mite (Tetranychus urticae) and mealybug species Phenacoccus solenopsis are the most prevalent pests found on this plant in India (Sharma et al. 2012; Sharma and Pati 2013). However, very few reports of begomoviruses infection on Withania somnifera have been found so far. Tobacco leaf curl virus (Phatak and Raychaudhuri 1967), Eggplant mottled dwarf virus (Al-Musa and Lockhart 2008) and Jatropha mosaic India virus (Baghel et al. 2012) have been reported to be infecting W. somnifera. Earlier reports were based on partial sequence and incomplete characterisation of the associated viruses and symptoms were quite different in contrast to present disease.

Typical symptoms like downward leaf curling, rolling, leaf deformation, vein clearing, and infestation of whiteflies were observed for the first time on the leaves of W. somnifera in the experimental fields of CSIR-Central Institute of Medicinal and Aromatic Plants (CIMAP), Lucknow, India. The identification and molecular characterization of the pathogen responsible for the disease associated with W. somnifera is described in the present study.

Materials and methods

Sample collection

During the survey, in October–November, 2019, W. somnifera leaves exhibiting characteristic viral symptoms were collected from the experimental field of CSIR-CIMAP, Lucknow, India. Ten symptomatic and three asymptomatic (healthy) samples were taken from different corners of the fields for downstream analysis.

Sap inoculation

Mechanical sap inoculation on healthy W. somnifera, Nicotiana benthamiana, N. glutinosa and Chenopodium amaranticolor seedlings were done using pre dusted carborundum leaves with the sap obtained by naturally infected leaf tissue ground in the presence of chilled phosphate buffer in an autoclaved pestle and mortar. Just after inoculation, leaves were gently washed with distilled water. Plants inoculated with phosphate buffer only served as mock control. All the plants were kept under glasshouse at 25 ± 2 °C for the development of symptoms.

Transmission electron microscopy

Healthy and infected leaf samples were processed for the electron microscopy as described by Garg et al. (2001), and preparations were negatively stained in 2% w/v aqueous uranyl acetate. The grids were dried and screened under a JEOL-100S transmission electron microscope at 80 kV.

Whitefly transmission

For transmission studies, healthy, starved indigenous B-biotype whiteflies (taken from CSIR-NBRI, Lucknow) were used for acquisition feeding (24 h) on infected W. somnifera leaves and then transferred to healthy plants (10/plant) for an overnight inoculation feeding. After inoculation feeding, whiteflies were killed by spraying mild (0.01%) insecticide/acaricide (OMITE-Propagite 57% EC). The plants were kept under glasshouse for further observations.

Molecular characterization

Total genomic DNA was isolated from symptomatic and asymptomatic leaves of W. somnifera using CTAB method as described by Khan et al. (2007). PCR was performed in 25 μl PCR mixture containing 2.5 μl 10X buffer, 0.8 μl of each primer (10 μM), 1 μl of 10 mM dNTPs, 0.8 μl of DNA templates (30 ng), 18.8 μl of Milli Q and 0.3 μl of Taq DNA polymerase (3 U/μl) using begomovirus CP-specific primers (Hallan et al. 1998) for initial screening of begomovirus infection in the respective samples. Amplified PCR products were processed for the whole genome amplification. Two sets of overlapping primers K1F/K1R and K2F/K2R (Kumar et al. 2011) and universal primers for the detection of DNA-B and alphasatellite components (Rojas et al. 1993) were used. Primer pair β01/β02 was used to detect the presence of betasatellite (Briddon et al. 2002). Amplified PCR products were cloned and sequenced in CSIR-CIMAP using ABA sequencer 3730 (Applied Biosystems).

Rolling Circle Amplification (RCA) was performed using (RCA, Templiphi GE Healthcare, USA) to amplify the full-length genome of the begomoviruses (Inoue-Nagata et al. 2004). The RCA product was digested independently with various restriction enzymes (BamHI, HincII, SalI, KpnI, PstI, and XbaI) to linearize full length genomic DNA. The restricted fragments were resolved on a 1% agarose gel.

Sequences were analysed using BLASTn and deposited to Genbank database. Phylogenetic analysis was carried out with representative sequences of begomoviruses available in the NCBI database using the Neighbour Joining method in MEGA 7 software with 1000 bootstrap value (Kumar et al. 1994).

Results and discussion

During the survey, infected plants showed typical severe downward leaf curling, rolling, vein clearing, wilting, leaf deformation and poor growth (Fig. 1). Based on the symptoms and heavy infestation of whiteflies, infection of begomoviruses was suspected. Mechanically inoculated W. somnifera and other plants did not express any visible local or systemic symptoms on the leaves and all were also found negative in PCR tests. Disease incidence was estimated about 17–20 per cent on the basis of visual plant population.

Fig. 1
figure 1

a Healthy plants of W. somnifera and b infected plants of W. somnifera exhibiting vein clearing, severe leaf curling and rolling symptoms in the field condition

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Partially purified virus preparations from the infected samples were observed under TEM; 10–15 windows were screened and revealed the presence of scattered twinned icosahedral particles of ~ 18–20 nm in diameter (Fig. 2).

Fig. 2
figure 2

TEM image showing twinned icosahedral virus particles (~ 18–20 nm in diameter) negatively stained with uranyl acetate in infected samples (Bar-100 nm)

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B. tabaci-based transmission was found positive in five of the eight Withania plants, and developed symptoms similar to those observed on the naturally infected plants in the fields.

PCR amplicon of ~ 771 bp was obtained using coat protein specific primers in eight out of ten symptomatic samples, whereas no amplification was obtained in asymptomatic samples. Samples from which CP gene was amplified were used for further detection of genomic and satellite components of begomoviruses. Genomic DNA-A fragment was amplified in two parts of ~ 1200 bp and ~ 1700 bp via PCR (Fig. 3) and find out the actual length of the DNA segment by removing excess bases. However, presences of DNA-B and alphasatellite components were not detected by PCR. Betasatellite of 1376 bp was detected using primers β01/β02. The presence of a begomovirus and associated betasatellite was also validated by rolling circle amplification. The RCA products restricted with HincII showed the presence of two fragments of 2.7 kb and 1.3 kb corresponding to DNA-A and betasatellite of begomovirus while BamHI, KpnI, PstI, SalI and XbaI generated 2.7 kb fragment only, suggesting these enzymes have single restriction site in DNA A and no restriction site in betasatellite (Fig. 4).

Fig. 3
figure 3

PCR amplification of the full genome of DNA-A using primers K1F/K1R and K2F/K2R generating two overlapping fragments of ~ 1.2 kb and ~ 1.7 kb, respectively, from infected W. somnifera samples. M- λ EcoRI/ Hind III DNA marker, Lanes 1 and 2—PCR products of ~ 1.2 kb and Lanes 3 and 4—PCR products of ~ 1.7 kb

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Fig. 4
figure 4

Restriction profile of RCA product (generated from DNA isolated from infected W. somnifera samples), digested with different restriction enzymes. M- λ EcoRI/ Hind III DNA marker, Lane 1—HincII, Lane 2—BamHI, Lane 3—KpnI, Lane 4—PstI, Lane 5—SalI and Lane 6—XbaI

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Sequences of DNA-A (2758 bp) and betasatellite (1347 bp) were deposited to NCBI Genbank database with the accession numbers OP617239 and OP617240, respectively. The genome organization is found to be of Old World monopartite begomoviruses consist of six ORF’s, two on virion sense strand and four on complementary strand, interspaced by an IR (Intergenic region). The viral sense strand has ORF AV1 (encoding CP) which is partially overlapped by the small ORF AV2 (Pre- CP), while the complementary sense strand contains ORFs AC1 (Rep), AC2 (TrAP), AC3 (REn), and AC4. BLASTn analysis of DNA-A has shown maximum similarity to Cherry tomato leaf curl virus (LN906594) with 88% sequence identity; suggesting it to be a novel begomovirus. Based on the nomenclature guidelines for Begomovirus (Brown et al. 2015), we concluded the present virus to be a novel begomovirus for which we proposed the name Withania leaf curl virus. Phylogenetic analysis revealed that since Withania leaf curl virus (Acc.no OP617239) is a new virus, its DNA-A sequence formed a separate clade and it shared a common ancestor with Cherry tomato leaf curl virus (LN906594) (Fig. 5). Betasatellite sequence showed 98% identity with Ageratum yellow vein betasatellite (KF364485) reported from Lucknow, India and shared the same clade of the phylogenetic tree (Fig. 6).

Fig. 5
figure 5

Phylogenetic relationship based on nucleotide sequence of DNA-A of begomovirus isolate (OP617239) infecting W. somnifera with different begomoviruses using MEGA 7.0 neighbour joining tree method with 1000 bootstrap value. Withania leaf curl virus (Acc.no OP617239) forms a separate clade and it shares a common ancestor with Cherry tomato leaf curl virus (LN906594)

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Fig. 6
figure 6

Phylogenetic relationship based on nucleotide sequence of betasatellite DNA of begomovirus isolate (OP617240) infecting W. somnifera with different begomoviruses using MEGA 7.0 neighbour joining tree method with 1000 bootstrap value. Betasatellite sequence has maximum identity with Ageratum yellow vein betasatellite (KF364485) reported from Lucknow, India, and shares the same clade in the phylogenetic tree

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The infection of W. somnifera by Tobacco leaf curl virus (Phatak and Raychaudhuri 1967), Eggplant mottled dwarf virus (Al-Musa and Lockhart 2008) and Jatropha mosaic India virus (Baghel et al. 2012) are the few reports with partial sequences and that have not been fully characterised are available in the literature. However, this is the first natural occurrence of a novel begomovirus, Withania leaf curl virus along with Ageratum yellow vein betasatellite associated with severe leaf curling, rolling and vein clearing on W. somnifera. It is one of important medicinal herb of versatile use and mostly people preferred to keep in the house, garden and also cultivated in the commercial fields. The present and earlier reported viruses are spreading in nature by vectors and causing significant yield reduction; therefore, their management strategies should be implemented using available findings to protect other economically important crops including medicinal and aromatic plant.