The initial inoculum of DoYMV was collected from dolichos plants at the experimental field of Indian Agricultural Research Institute (IARI), New Delhi, India. The virus culture was established on dolichos plants (var. Pusa Garima) by B. tabaci inoculation and maintained under insect-proof conditions. The virus isolate was characterized based on the nucleotide sequence of DNA-A as described later.

An isofemale, virus-free line of B. tabaci Asia II 1 maintained for more than 5 years at Insect Vector Laboratory, Advanced Centre for Plant Virology, IARI was used in this study. The identity of B. tabaci was substantiated by sequencing mitochondrial cytochrome oxidase subunit I (mtCOI). Representative individuals of the population were randomly tested in PCR with generic begomovirus primers (Roy et al., 2015) to confirm its aviruliferous status. The freshly emerged adult females were collected using an aspirator for further experiments.

Total DNA was extracted by using cetyltrimethylammonium bromide (CTAB) methods with modifications (Rehman et al., 2021). In brief, CTAB extraction buffer was prepared in a total volume of 1 ml containing 100 mM Tris–HCl (pH 8.0), 1.4 M NaCl, 20 mM EDTA (pH 8.0), 2% CTAB, and 2 μl β-mercaptoethanol. Individual B. tabaci was crushed in 100 μl of CTAB extraction buffer inside a microfuge tube using a homogenizer. About 100 mg of plant tissue was crushed in 500 μl of extraction buffer using mortar–pestle. The lysate was vortexed and incubated at 65°C for 30 min. An equal volume of chloroform/isoamyl alcohol (24:1) was added to the lysate and centrifuged at 16,000 × g for 15 min. The upper aqueous phase was transferred to a fresh microfuge tube. The DNA was precipitated by adding 0.7 vol of ice-cold isopropanol and kept at −20°C for 1 h. The sample was centrifuged at 16,000 × g for 15 min and the supernatant was decanted gently. The pellet if any was gently washed with 100 μl of 70% ethanol. The ethanol was decanted and residual ethanol was removed by drying at room temperature. The pellet was dissolved in 20 μl sterile distilled water in the case of B. tabaci and 50 μl in the case of the plant. The concentration and purity of DNA were assessed in a spectrophotometer (NanoDrop One; Thermo Fischer Scientific, United States).

A partial fragment of the mtCOI gene of B. tabaci was amplified using primer pairs C1-J-2195 and TL2-N-3014 (Simon et al., 1994). PCR was carried out in a 25-μl reaction mixture containing 12.5 μl of 2 × PCR Master Mix (Thermo Fischer Scientific), 0.4 μM of each forward and reverse primer, 8.5 μl of nuclease-free water, and 2 μl (∼50 ng) of DNA template. The PCR was performed in a T100 Thermal Cycler (Bio-Rad, United States) as described by Rehman et al. (2021) and resolved on an agarose gel stained with GoodView (BR Biochem, India). The purified PCR product was sequenced bi-directionally and sequences were processed by BioEdit and BLASTn. A consensus sequence was submitted in the GenBank. A Bayesian Inference (BI) phylogeny was undertaken using MrBayes 3.2 considering a genetic divergence cutoff of 4% to confirm the cryptic species identity.

Initially, begomovirus infection in the dolichos plants collected from the experimental field was confirmed by PCR with generic degenerate primers for begomoviruses, GEM-F and GEM-R targeting DNA-A (Roy et al., 2015). PCR-positive samples were subjected to rolling cycle amplification (RCA) to amplify the circular DNA of the begomoviruses. A 20-μl RCA reaction was prepared using 10 ng template DNA, 0.2 mM dNTPs, 7 U Phi29 DNA polymerase (Thermo Fischer Scientific), 2 × exo-resistant random primer (Thermo Fischer Scientific), 0.4 U pyrophosphatase (Thermo Fischer Scientific), and 1 × Phi29 DNA polymerase buffer. RCA was performed at 65°C in a water bath for 18 h. The RCA products were linearized by restriction digestion with EcoR1. The linearized RCA products were ligated into a pUC18 vector and transformed into DH5α Escherichia coli cells. Plasmid DNA was extracted using Wizard Plus SV Minipreps DNA Purification System (Promega, United States) and sequenced. The sequences were processed by BioEdit and BLASTn was performed to check the species homology.

A pair of DoMYV-specific primers (AG223F and AG224R, Table 1) was designed for the detection of DoYMV in PCR and real-time PCR. All the nucleotide sequences of DoYMV available in NCBI were aligned in ClustalW and primers were designed from the conserved regions in the coat protein (CP) using Primer 3 Input version 0.4.0. The major aspects such as primer length, amplicon size, GC contents, intra-primer, and inter-primer homology were considered for primer designing. The site specificity of the primers was checked in Primer-BLAST¹. The newly designed primers were validated in a gradient PCR. Each 25-μl PCR reaction contained 12.5 μl of 2 × PCR Master Mix (Thermo Fischer Scientific), 0.4 μM of each forward and reverse primer, 8.5 μl of nuclease-free water, and 2 μl of DNA template (∼50 ng for B. tabaci and ∼100 ng for plants). The PCR was performed in a T100 Thermal Cycler with one cycle of initial denaturation at 95°C for 5 min, 30 cycles of denaturation at 95°C for 30 s, annealing at a temperature gradient of 50–55°C for 30 s, and extension at 72°C for 45 s followed by a final extension at 72°C for 10 min. The PCR products were resolved on 2% agarose gel stained with GoodView and visualized in a gel documentation system with a 100 bp plus DNA ladder. The annealing temperature that produced the best amplification without any secondary amplification was considered for detection of DoYMV in PCR and real-time PCR.

Real-time PCR was performed in a 48-well Insta Q48M (Himedia, India). A 20-μl reaction mixture consisted of a 10 μl 2 × DyNAmo ColorFlash SYBR Green qPCR Mix (Thermo Fischer Scientific), 2 μl template DNA (∼50 ng for B. tabaci and ∼100 ng for plant), and 0.5 μM of each forward and reverse primer. Thermal cycling was performed with one cycle of initial denaturation at 95°C for 5 min, 30 cycles of denaturation at 95°C for 30 s, annealing at 53°C for 30 s, and extension at 72°C for 45 s. Since SYBR Green I dye binds non-specifically to any double-stranded DNA, dissociation or melting stage was carried out after every reaction to determine the specificity of the amplification. Each of the biological replicates had three technical replicates. A no-template control (NTC) and positive control were included in each assay. The C_T values thus obtained were used to calculate the mean C_T and SEM using Microsoft Excel software.

Freshly emerged aviruliferous B. tabaci adult females were allowed to feed on a DoYMV-infected dolichos twig for 48 h of virus acquisition. Ten sets were maintained for virus acquisition, each containing around 50 adult flies. A few adults were randomly collected and tested in PCR as described previously to confirm the virus acquisition. The viruliferous adults were used to examine the transovarial transmission. All experiments were conducted in an insect-proof growth chamber at 27 ± 2°C, 60 ± 10% relative humidity, and 16 h light–8 h dark photoperiod.

Viruliferous adult females of B. tabaci were collected in groups of 10 flies from each virus acquisition set. Each group of viruliferous flies was released on 4-leaf stage eggplants (var. Navkiran, Mahyco), non-host for DoYMV, and covered with an insect breeding box (SPL Life Sciences, Korea) with nets at two sides and top. All the sets were kept in an insect-proof growth chamber at 27 ± 2°C, 60 ± 10% relative humidity, and 16 h light–8 h dark for egg-laying. The adult flies were shifted to a fresh eggplant daily for up to 10 days.

To determine the pattern of transovarial transmission of DoYMV in next-generation (F1) eggs, the eggs laid by the viruliferous parent B. tabaci each day on eggplants were collected separately in 10 biological replicates. Total DNA was isolated from a group of eggs day-wise and tested in PCR with DoYMV-specific primers as described previously.

To assess the frequency of transovarial transmission of DoYMV in F1 eggs, approximately 10 eggs from each day laying were randomly collected using sterile needles. All the eggs were washed in 70% ethanol to eliminate surface contaminations if any. Total DNA was extracted from individual eggs and tested in real-time PCR for the presence of DoYMV as described previously. Ten biological replicates with three technical replicates were tested. The percent of eggs infected by DoYMV in each biological replicate was calculated as the number of eggs found positive out of 10 eggs tested in real-time PCR.

To determine the pattern of DoYMV infection in F1 adults, F1 eggs laid by viruliferous parents on eggplants were incubated at 27 ± 2°C, 60 ± 10% relative humidity, and 16 h light–8 h dark. The freshly emerged F1 adults were collected separately from each day laying. Total DNA was isolated from each set of adult flies and the presence of DoYMV was tested in PCR as described previously. Ten biological replicates of adult flies were tested. The eggplant on which eggs were laid and the adults were raised, was also tested in PCR to confirm the absence of DoYMV.

The proportion of F1 adults carrying the virus was estimated by randomly collecting 10 freshly emerged adult flies from each day laying. Total DNA was isolated from individual adults. The presence of DoYMV was detected in individual adults by real-time PCR as described previously with 10 biological and three technical replicates. The proportion of F1 adults carrying DoYMV DNA was expressed as a percentage. A t-test was performed to determine the significant difference among the mean proportion of eggs and flies carrying DoYMV. Mean differences among the categories were separated by Tukey’s test at a CI of 95% using XLSTAT 2014.5.03.

Based on the results of detection of DoYMV from each day laying, a portion of F1 eggs laid in between 3 and 6 days post virus acquisition was used for generating next-generation adults (F1). The freshly emerged F1 adults were released on healthy eggplants (non-host) for egg-laying in five biological replicates. The second-generation eggs (F2) were collected from eggplants and tested in PCR for the presence of DoYMV as described previously. The host plant on which eggs were laid was also tested to confirm the absence of the virus.

Based on the results of detection of DoYMV in F1 adults, the adults that emerged from eggs laid in between 3 and 6 days post virus acquisition were used for the biological transmission of DoYMV in dolichos plants. Healthy dolichos plants (var. Pusa Garima) were raised in an insect-proof plant growth chamber. All the plants were tested in PCR before DoYMV inoculation to confirm their healthy status. The F1 adults were released on healthy dolichos plants at a 4-leaf stage for 48 h of inoculation feeding. Fifteen plants were inoculated in three replicates. Ten adult flies per plant were used for inoculation. An equal number of plants were mock inoculated with aviruliferous B. tabaci that were not exposed to DoYMV. The inoculated plants were maintained under insect-proof conditions at 27 ± 2°C, 60 ± 10% relative humidity, and 16 h light–8 h dark photoperiod. All the plants were regularly monitored for symptom development and tested in PCR for infection of DoYMV at 15 days post inoculation.

To understand the localization of DoYMV in B. tabaci, a tetramethylindo(di)-carbocyanine (Cy3)-conjugated fluore- scence oligonucleotide probe AG223F-Cy3 (Table 1) was used. The probe was conjugated with Cy3 at its 5’ end complementary to 20 nucleotide sequences in the viral strand of DoYMV CP. The invasion of DoYMV in the reproductive system and eggs of B. tabaci was localized using the Cy3-probe. The ovaries of DoYMV-exposed B. tabaci parents were dissected post 48 h of virus acquisition under a stereomicroscope. The eggs laid on 4-day post DoYMV acquisition by parent B. tabaci were used for DoYMV localization. The hybridization procedure was followed as described by Ghanim et al. (2009) with modifications. The specimens were washed several times with sterile water and fixed by using Carnoy’s fixative (chloroform/methanol/glacial acetic acid, 6:3:1, v/v) for 2 h at room temperature. The specimens were washed thrice with a hybridization buffer containing 20 mM Tris–HCl (pH 8.0), 0.9 M NaCl, 0.01% sodium dodecyl sulfate (SDS), and 30% urea. The hybridization was performed overnight with Cy3-probe at 10 pmol/ml in hybridization buffer. The specimens were washed three times with hybridization buffer and mounted on a glass slide. The Cy3-labeled specimens were observed under a confocal laser scanning microscope (TCS SP5 II; Leica, Germany). The photographs were captured and processed in LAS AF software (Leica). Ovaries and eggs of aviruliferous B. tabaci not exposed to DoYMV were taken as untreated control.

For preparing a standard curve of DoYMV, the PCR-amplified product of DoYMV using primer pair AG223F and AG224R was ligated in a pJET1.2 vector using CloneJET PCR Cloning Kit (Thermo Fischer Scientific) and transformed into DH5α E. coli cells. A 10-fold serial dilution of the linearized plasmid DNA from 1 to 10^–5 ng was used as a template in real-time PCR with three technical replicates. Real-time PCR was performed as described previously. Each reaction was followed by the dissociation or melting stage. The standard curve for DoYMV was prepared by plotting a linear regression curve with the mean C_T values on Y-axis and log DNA dilution (ng) on X-axis.

Virus titer present in eggs, third instar nymphs, freshly emerged adults, and 10-day-old adults of next-generation B. tabaci post virus acquisition by parents were assessed in real-time PCR. DNA was isolated from individual B. tabaci and used as a template in real-time PCR. Real-time PCR was performed with primer pair AG223F and AG224R as described previously. The absolute quantification of DoYMV present in individual B. tabaci egg, third instar nymph, freshly emerged, and 10-day-old adults was carried out by fitting the mean C_T values in the standard curve. DoYMV titer calculated in nanograms using the standard curve was converted into the copy number of DoYMV using the following formula. N = (X ng × 6.0221 × 10²³ molecules/mole)/(n × 330 g/mol × 10⁹ ng/g), where N is the number of viral copies, X is the amount of amplicon (ng), and n is base pairs of recombinant plasmids. The average copy numbers of DoYMV were calculated along with standard error out of three biological and three technical replicates. ANOVA was used to test the significant difference among the virus copies in different life stages of B. tabaci. Mean differences among the categories were separated by Tukey’s test at a CI of 95% using XLSTAT 2014.5.03. The increase and decrease in DoYMV copies in different life stages of B. tabaci were compared to understand the virus propagation.

PCR with primer pair C1-J-2195 and L2-N-3014 produced an expected amplicon of ∼860 bp of B. tabaci mtCOI as visualized on 1% agarose gel (data not shown). The consensus nucleotide sequence exhibited about 99.99% identity with other B. tabaci sequences in NCBI upon BLASTn analysis. The sequence is available in GenBank with accession no. MT920041. In BI phylogeny with 4% genetic divergence cutoff, the present population clustered with other B. tabaci Asia II 1 cryptic species.

Linearization of the RCA product of begomovirus positive sample by EcoR1 digestion produced ∼2.7-kb amplicon on 1% agarose gel. The identity of the virus was confirmed by sequencing the cloned RCA product. Bidirectional sequencing of the cloned product produced 2,761 nt sequence comprising complete DNA-A that showed nearly 98.19% homology to other DoYMV isolates in BLASTn analysis. The sequence can be retrieved by GenBank accession no. MZ821026. The primer pair, AG223F and AG224R, targeting DoYMV CP produced ∼180 bp sharp band in gradient PCR at annealing temperature 53–55°C. Based on the quality of amplification, annealing temperature was standardized at 53°C for PCR and real-time PCR assays. The nucleotide sequences of the amplicon (GenBank accession no. MZ503593) showed 96% homology with other DoYMV isolates.

The transovarial transmission of DoYMV was detected in PCR by testing a group of eggs laid at different days post virus acquisition. The eggs laid immediately after the virus exposure were free from DoYMV viral DNA. The first invasion of DoYMV in eggs was detected 3 days post virus acquisition (Figure 1). Eggs laid in between 3 and 6 days post virus acquisition were DoYMV positive in PCR. The presence of DoYMV DNA was not recorded beyond 6 days post acquisition. The virus titer in individual eggs beyond 6 days might either be too low to detect in PCR or not present at all. An increase in DoYMV exposure to B. tabaci parents extended the virus passage in F1 eggs beyond 6 days.

Ten randomly selected individual eggs laid by the DoYMV-exposed parents from each of 10 virus acquisition sets were tested in real-time PCR. The real-time PCR further substantiated the PCR results. No virus was detected in individual eggs before 3 days and after 7 days post DoYMV acquisition by parent B. tabaci. About 51% of the eggs laid on 3 days post virus acquisition were DoYMV positive in real-time PCR (Figure 1). An almost equivalent proportion (53%) of eggs laid on 4 days post acquisition carried DoYMV viral DNA. The proportion of DoYMV-positive eggs dropped slightly on 5 to 6 days post acquisition to 31%. A single specific peak at 81°C in melt curve analysis indicated specificity of the reactions.

A similar trend of DoYMV infection was recorded in F1 adults. F1 adults emerged from the eggs around 3 weeks post oviposition. Only the F1 adults generated from eggs that were laid by DoYMV-exposed parents in between 3 and 6 days post virus acquisition were DoYMV positive in PCR (Figure 1). F1 adults emerged from eggs laid before 3 days and after 7 days post virus acquisition by parent B. tabaci were free from DoYMV infection. About 27–30% of adults that emerged from eggs laid on 3–4 days post acquisition were found DoYMV positive in real-time PCR. The proportion of F1 adults with DoYMV DNA raised to 37% on 5–6 days post virus acquisition. A single peak specific to DoYMV in the melt curve analysis of real-time PCR indicated the specificity of reactions. DoYMV was not detected in F1 adults that emerged from eggs laid 7 days onward. A slight decrease in the proportion of virus infection in adults than eggs might be due to the fall of virus titer in individual adults beyond the detection limit of real-time PCR.

The F2 eggs laid by the DoYMV-positive F1 adults were also assessed to understand the passage of DoYMV from the first to second generation of B. tabaci progenies. F2 eggs laid by DoYMV-positive F1 adults retained the DoYMV viral DNA as detected in PCR (Figure 1). This was indicative of the passage of DoYMV beyond the first-generation progenies of B. tabaci. The eggplant on which the next-generation B. tabaci was reared, had been found DoYMV negative in PCR.

To assess the infectivity of DoYMV-positive F1 adults, biological transmission was conducted. F1 adults emerged from eggs laid in between 3 and 6 days post virus acquisition by viruliferous parent B. tabaci, transmitted DoYMV successfully to healthy dolichos plants. All the test plants inoculated by F1 adults were found positive to DoYMV in PCR at 15 days post inoculation. The characteristic bright yellow mosaic symptoms appeared at 21–25 days post inoculation (Figure 2). Plants that were mock inoculated by aviruliferous B. tabaci were found PCR negative and did not produce any symptoms.

The presence of DoYMV was localized in ovaries and eggs of B. tabaci adult females post virus exposure using a Cy3-probe. The specimens were washed until no viral DNA was detected by PCR from the wash to avoid contaminations from the hemolymph. Strong Cy3-specific fluorescence was observed in ovaries of B. tabaci post 48 h of exposure to DoYMV (Figure 3). The presence of fluorescence in ovaries of B. tabaci indicated that DoYMV invaded the reproductive system of B. tabaci. Localization of DoYMV was also distinguished by Cy3-fluorescence in the eggs laid by the DoYMV-exposed B. tabaci parents. The results were consistent with the detection of DoYMV in eggs by PCR and real-time PCR. No corresponding fluorescence signal was observed in ovaries and eggs of aviruliferous B. tabaci that indicated the specificity of the assay.

Virus titer was quantified in eggs, third instar nymphs, freshly emerged adults, and 10-day-old adults of F1 progeny. The F1 progeny developed from the eggs laid on 4 days post acquisition was considered for this study. The virus titer started increasing during the nymphal instars. A 1.68-fold increase in the virus titer was recorded in the third instar B. tabaci compared with the egg (Figure 4). The virus titer estimated in nanograms using the standard curve in real-time PCR was converted into a copy number profile. The standard curve of DoYMV (Figure 4) showed high amplification efficiency of 93.15% indicating acceptable conditions for absolute quantification. The standard curve showed a coefficient of correlation (R²) of 1. The amplicon produced a specific peak at 81°C without any secondary peak in melt curve analysis, indicating the specificity of reaction. The eggs laid on 4 days post virus acquisition by B. tabaci parents contained on an average 1.43E + 15 copies of DoYMV, whereas it was 2.38E + 15 copies in third instar nymphs. The virus titer dropped down in freshly emerged adults and was almost equivalent to eggs. The virus copies were further reduced to 7.68E + 4 copies in 10-day-old adults. An increase in DoYMV copies during nymphal instars indicated its propagation in B. tabaci for a brief period. The propagation might be for a short period and started declining in adult stage.