Genetic diversity and characterization of cucumber mosaic virus infecting chilli in Zone II and Zone IIIB of Bihar, India

Chilli (Capsicum annuum L.) is an economically important spice and vegetable crop widely cultivated in Bihar; however, its productivity is severely affected by viral diseases, particularly cucumber mosaic virus (CMV). The present study was undertaken to investigate the occurrence, molecular characterization, and genetic diversity of CMV infecting chilli in Zone II and Zone IIIB of Bihar. Extensive field surveys conducted during 2019–2020 in major chilli-growing districts, including Kishanganj, Purnia, and Bhagalpur, revealed characteristic symptoms such as mosaic, mottling, chlorosis, leaf deformation, vein clearing, shoestring leaves, and stunted growth. A total of 355 symptomatic samples were collected and screened through Direct Antigen Coating Enzyme-Linked Immunosorbent Assay (DAC-ELISA), among which 115 samples tested positive for CMV infection. Representative isolates from different locations were biologically maintained through mechanical sap inoculation on indicator hosts. Total RNA extracted from infected plants was subjected to reverse transcription–polymerase chain reaction (RT-PCR) using coat protein (CP) gene-specific primers, which amplified the expected 657 bp fragment from six selected isolates, namely Sabour, Kahalgoan, Purnia, Kasba, Belwa, and Kharkhari. The amplified products were cloned, sequenced, and submitted to GenBank. Comparative nucleotide and deduced amino acid sequence analyses revealed a high degree of sequence conservation among the Bihar isolates, with only minor nucleotide polymorphisms and limited amino acid substitutions. Phylogenetic analysis based on CP gene sequences grouped all six Bihar isolates within CMV subgroup IA, indicating close genetic relatedness with previously reported Indian CMV isolates. The Sabour and Kahalgoan isolates formed a closely related cluster, whereas the remaining isolates also grouped within the same lineage with minimal genetic divergence. The observed sequence conservation suggests that CMV populations infecting chilli in Bihar are genetically stable and may have originated from a common ancestral source followed by regional dissemination. The present study provides a comprehensive molecular characterization and phylogenetic analysis of CMV infecting chilli in Zone II and Zone IIIB of Bihar. The findings highlight the importance of continuous surveillance, molecular diagnostics, and the development of resistant cultivars for effective management of CMV-associated diseases and sustainable chilli production.

Chilli (Capsicum annuum L.) is an economically important spice and vegetable crop cultivated widely in tropical and subtropical regions. India is among the leading producers and exporters of chilli, where the crop contributes substantially to agricultural income, food industries, and rural livelihoods (1, 2). However, chilli productivity is severely constrained by several biotic stresses, particularly viral diseases that significantly reduce yield and fruit quality. More than thirty viruses have been reported to infect chilli worldwide, among which cucumber mosaic virus (CMV) is considered one of the most destructive because of its wide host range, efficient transmission, and extensive genetic variability (3, 4).

CMV, a member of the genus Cucumovirus under the family Bromoviridae, infects over 1,000 plant species including vegetables, ornamentals, pulses, and weeds (5). The virus is transmitted mainly by aphids in a non-persistent manner and survives in alternate hosts and weed reservoirs, facilitating rapid dissemination under field conditions (5, 6). Seed transmission has also been reported in several hosts, contributing to long-distance spread and disease establishment in new cultivation areas (7). Recent studies further demonstrated that CMV infection can alter host volatile emissions and plant physiology, thereby enhancing aphid-mediated transmission efficiency and epidemiological fitness of the virus (8).

The CMV genome consists of three positive-sense single-stranded RNA molecules designated as RNA1, RNA2, and RNA3, which encode proteins involved in replication, movement, pathogenicity, and suppression of host defense responses (5). Molecular studies have revealed that interactions between viral proteins and host cellular factors play an important role in symptom development, chlorosis, and systemic infection (9). Based on serological properties, nucleotide divergence, and phylogenetic relationships, CMV isolates are classified into subgroup I and subgroup II, with subgroup I further divided into IA and IB lineages (10). Previous studies from India have shown that most CMV isolates infecting chilli and other crop hosts predominantly belong to subgroup IA (11–13). However, the majority of these investigations were confined to southern, western, or central India, while molecular epidemiological information from eastern India, particularly Bihar, remains very limited.

The coat protein (CP) gene is widely used for molecular characterization and phylogenetic analysis of CMV because it plays a crucial role in virus encapsidation, vector transmission, host interaction, and viral evolution (14). Sequence analysis of the CP gene provides valuable information regarding genetic diversity, subgroup classification, evolutionary relationships, and geographical distribution of CMV isolates. Recent molecular and high-throughput sequencing studies have demonstrated substantial nucleotide variability among global CMV populations resulting from mutation, recombination, and genomic reassortment, enabling adaptation to diverse hosts and ecological conditions (15–17). Such evolutionary plasticity may lead to the emergence of genetically distinct strains with altered pathogenicity and symptom expression, thereby complicating disease diagnosis and management.

Bihar represents an important emerging chilli-growing region where viral diseases are increasingly becoming a major constraint to sustainable production. Nevertheless, systematic information regarding the occurrence, molecular diversity, and phylogenetic relationships of CMV infecting chilli in Zone II and Zone IIIB of Bihar has not been comprehensively documented. Earlier Indian studies mainly reported subgroup affiliation without extensive regional sampling or comparative phylogenetic evaluation associated specifically with chilli ecosystems of eastern India. Since ecological conditions, cropping systems, host diversity, and vector prevalence can influence the evolution and dissemination of CMV populations, region-specific molecular characterization is essential for understanding local virus epidemiology and disease dynamics.

Therefore, the present investigation was undertaken to study the occurrence, molecular characterization, and genetic diversity of CMV infecting chilli in Zone II and Zone IIIB of Bihar. The study further aimed to analyze nucleotide and amino acid variability of coat protein gene sequences and determine the phylogenetic relationships of Bihar isolates with previously reported Indian and global CMV isolates. The findings provide the first comprehensive molecular evidence confirming the predominance of CMV subgroup IA in chilli from these agro-climatic regions of Bihar and establish a baseline dataset for future surveillance, molecular diagnostics, and integrated disease-management strategies.

Field surveys were systematically conducted during 2019 and 2020 in the chilli-growing regions of Zone II and Zone IIIB of Bihar to investigate the occurrence and distribution of viral diseases affecting chilli cultivation. Initial diagnosis based solely on field symptomatology was not definitive, since several chilli-infecting viruses induce similar and overlapping symptoms under natural conditions. Nevertheless, plants infected with CMV commonly exhibit distinct symptoms such as mosaic and mottling, chlorosis, vein clearing, leaf deformation, shoestring-like narrowing of leaves, reduction in plant vigor, stunted growth, and whitish streaking or discoloration on fruits (18).

During the present investigation, chilli plants expressing suspected viral symptoms were collected from representative locations of Zone II and Zone IIIB of Bihar. In Zone II, extensive sampling was undertaken in Kishanganj district from Kharkhari, Motihara, Ganiyabari, Lodhabari, and Arrabari. Additional symptomatic samples were also collected from Purnia district, including Kasba, Jalalgarh, and Dagarua. In Zone IIIB, surveys were conducted in Bhagalpur district, particularly in Sabour and Kahalgoan. Sampling was performed on the basis of visible symptoms associated with viral infection, with special emphasis on symptom complexes suggestive of CMV infection. The collected samples were subsequently utilized for DAC-ELISA confirmation and molecular characterization of the virus.

Virus culture maintenance and symptomatological studies

Preliminary detection and confirmation of CMV in collected chilli samples were carried out using Direct Antigen Coating Enzyme-Linked Immunosorbent Assay (DAC-ELISA) and reverse transcription polymerase chain reaction (RT-PCR) with CMV coat protein gene-specific primers. Based on serological and molecular confirmation, six representative CMV isolates from Bihar were selected for further studies. These included four isolates from Zone II (Belwa-chilli, Kharkhari-chilli, Purnia-chilli, and Kasba-chilli) and two isolates from Zone IIIB [Sabour-chilli and Kahalgoan-chilli]. The selected isolates were mechanically inoculated and subsequently maintained on cowpea (Vigna unguiculata) and Chenopodium album, which served as indicator and maintenance hosts for subsequent experiments.

For maintenance of virus cultures, healthy seedlings of cowpea and chilli at the two-leaf stage were raised under insect-proof greenhouse conditions to prevent accidental vector-mediated contamination. Infected chilli leaf tissues were homogenized in chilled phosphate buffer (pH 7.0) containing 0.02% β-mercaptoethanol. Carborundum powder (600 mesh) was applied to the leaf surface as an abrasive to facilitate virus entry during mechanical inoculation. The prepared sap extract was gently rubbed onto the leaves of test plants following the standard leaf abrasion method (19).

After inoculation, plants were maintained in a greenhouse at 30 ± 2 °C and regularly monitored for symptom development. Observations included local lesion formation, mosaic symptoms, chlorosis, leaf deformation, and systemic infection responses depending on the host species. Successful transmission and establishment of CMV infection in inoculated plants were further validated through DAC-ELISA and RT-PCR assays. These maintained cultures were used as a reliable source of inoculum for symptomatological, molecular, and phylogenetic studies.

Direct antigen coating–ELISA

Direct Antigen Coating Enzyme-Linked Immunosorbent Assay (DAC–ELISA) was performed following the method of Hobbs et al. (20) with slight modifications, for serological detection of CMV. Leaf tissues from test samples, along with positive and negative controls, were homogenized in coating buffer at dilutions of 1:1, 1:20, and 1:50. The extracts were centrifuged at 10,000 rpm for 10 min to obtain clarified sap. A volume of 100 µl of the supernatant was added to each well of a polystyrene ELISA plate and incubated at 37 °C for 3 h for antigen adsorption. Plates were washed three times with PBS-Tween buffer after each step. Wells were then blocked with 100 µl blocking buffer and incubated at 37 °C for 3 h to minimize non-specific binding. Subsequently, 100 µl of CMV polyclonal antiserum diluted 1:1000 was added to each well, followed by incubation. After washing, 100 µl of goat anti-rabbit alkaline phosphatase-conjugated secondary antibody (Sigma-Aldrich), diluted 1:5000, was added and incubated under similar conditions. Following final washing, 100 µl of p -nitrophenyl phosphate substrate solution was added to each well and incubated in the dark for 15–20 min for color development. Absorbance was recorded at 405 nm using a microplate reader (BioTek), and samples showing significantly higher absorbance than healthy controls were considered CMV positive. ELISA of each test sample was carried out in three replicate and mean of OD calculated for three replications.

RNA isolation, cDNA synthesis, and RT-PCR amplification

Total RNA was extracted from symptomatic and healthy leaves of cowpea (Vigna unguiculata L.) using the GeneJET Plant RNA Purification Mini Kit (Thermo Scientific, Cat. No. K0801) according to the manufacturer’s protocol. The quality and integrity of isolated RNA were assessed spectrophotometrically and used for complementary DNA (cDNA) synthesis. First-strand cDNA synthesis was carried out using a commercial reverse transcription kit (HiMedia, India). Briefly, approximately 100 ng of total RNA was mixed with gene-specific reverse primer and nuclease-free water to the desired reaction volume. The reaction mixture was denatured at 65 °C for 2 min and immediately chilled on ice. Subsequently, reverse transcription master mix containing reaction buffer, dithiothreitol (DTT), dNTPs, and M-MLV reverse transcriptase was added, followed by incubation at 37 °C for 90 min. The reverse transcriptase enzyme was inactivated at 85 °C for 5 min, and the synthesized cDNA was stored at −20 °C until further use.

Primers specific to the coat protein (CP) gene of CMV were designed using Primer3 software. Primer specificity was validated by multiple sequence alignment of representative CMV isolates retrieved from the NCBI database using CLUSTALW, and primer melting temperatures were analyzed using the IDT OligoAnalyzer tool.

PCR amplification was performed in a 25 µl reaction mixture containing 1× PCR buffer, dNTP mix, 0.5 µM each of forward and reverse primers, 1 U Taq DNA polymerase, and cDNA template. Thermal cycling was carried out with an initial denaturation at 94 °C for 4 min, followed by 30 cycles of denaturation at 94 °C for 50 s, primer-specific annealing, and extension at 72 °C. A final extension step was performed at 72 °C for 10 min. The amplified PCR products were resolved by electrophoresis on 1% agarose gel, stained with ethidium bromide, and visualized using a UV gel documentation system.

Cloning and sequencing of the coat protein gene

The amplified CP gene fragments were excised from agarose gel under UV illumination using a sterile scalpel and purified using a gel extraction procedure. Briefly, gel slices were dissolved in solubilization buffer at a ratio of 1:3 (w/v) and incubated at 55 °C until complete dissolution. An equal volume of isopropanol was added, and the mixture was transferred onto a silica-based spin column followed by centrifugation at 6000 rpm for 1 min to facilitate DNA binding. The column was subsequently washed with 750 µl wash buffer, followed by an additional centrifugation step at 10,000 rpm for 1 min to remove residual ethanol. Purified DNA was eluted in 40 µl of 10 mM Tris-HCl buffer (pH 8.0).

The purified CP gene amplicons were ligated into the pGEM-T Easy vector system (Promega, USA) according to the manufacturer’s instructions. Ligation reactions were prepared using an approximately 3:1 molar ratio of insert DNA to vector DNA and incubated with T4 DNA ligase at 4 °C for 6 h. The ligation products were transformed into competent Escherichia coli cells by standard transformation procedures. Putative recombinant colonies containing the CMV CP gene insert were screened by colony PCR and further confirmed through restriction enzyme digestion analysis. Confirmed recombinant plasmids were subsequently used for nucleotide sequencing and downstream molecular characterization.

Sequencing and phylogenetic analysis

Recombinant plasmids confirmed by restriction enzyme digestion were purified using a plasmid extraction kit and submitted for bidirectional sequencing. Sequencing reactions were performed using universal primers (T7, SP6, and M13 forward/reverse) as well as gene-specific primers to ensure complete and accurate coverage of the inserted coat protein (CP) gene fragment.

The obtained nucleotide sequences were edited, trimmed, and assembled into consensus sequences using BioEdit software. Final sequences in FASTA format were subjected to similarity searches using the BLASTn algorithm against the NCBI non-redundant nucleotide database to confirm virus identity. Sequence matches were evaluated on the basis of E-value, percentage query coverage, and nucleotide identity. Putative restriction enzyme recognition sites were analyzed using NEBcutter.

For comparative analysis, representative CMV coat protein gene sequences from India and other countries were retrieved from GenBank and aligned with the present isolates. Multiple sequence alignment was performed using the MUSCLE algorithm implemented in MEGA version 12. Phylogenetic relationships were inferred using the Neighbor-Joining (NJ) method based on evolutionary distance matrices generated by the Kimura two-parameter model. The reliability of tree topology was assessed through bootstrap analysis with 1,000 replications. Branches supported by higher bootstrap values were considered robust, and the resulting dendrogram was used to determine genetic relatedness, subgroup clustering, and evolutionary relationships among CMV isolates. Pairwise nucleotide identity matrices were also generated in MEGA 12 and further cross-validated through BLASTn analysis.

Field survey and sample collection

Two field surveys were conducted during 2019 and 2020 and number of samples collected from each survey from concerned district is mentioned in Table 1. The revealed the widespread occurrence of virus-like diseases in chilli-growing regions of agro-climatic Zones II and IIIB of Bihar. Since chilli cultivation in these areas is predominantly practiced on small and fragmented holdings for household consumption and supplemental income, survey efforts were focused on multiple scattered production pockets rather than on large continuous commercial fields. Symptomatic plants were identified and sampled on the basis of visible foliar and fruit symptoms suggestive of viral infection.

In Zone II, surveys were undertaken in Kishanganj district at Kharkhari, Motihara, Ganiyabari, Lodhabari, and Arrabari, and in Purnia district at Kasba, Jalalgarh, and Dagarua. In Zone IIIB, surveys were conducted in the major vegetable-growing localities of Sabour and Kahalgoan in Bhagalpur district. Plants selected for sampling exhibited characteristic symptoms commonly associated with CMV, including mosaic, mottling, chlorosis, vein clearing, leaf distortion, shoestring (filiform) leaves, stunted growth, and occasional whitish streaking on fruits (Figure 1).

Field diagnosis based solely on symptoms was difficult because CMV symptoms frequently overlapped with those induced by other chilli-infecting viruses such as Chilli veinal mottle virus (ChiVMV) and Capsicum chlorosis virus (CaCV). However, Chilli leaf curl virus (ChiLCV), which induces typical upward leaf curling, puckering, and reduced leaf lamina, could be visually differentiated and was excluded from the present CMV-oriented investigation.

A total of 165 symptomatic samples were collected from different chilli-growing locations of Kishanganj district, including Kharkhari, Motihara, Ganiyabari, Lodhabari, and Arrabari. In addition, 70 symptomatic samples were collected from the surveyed areas of Purnia district, namely Kasba, Jalalgarh, and Dagarua. From the vegetable-growing belt of Bhagalpur district, particularly Sabour and Kahalgoan located near the Ganga River, 95 symptomatic samples were collected. Overall, 355 symptomatic chilli samples were collected during the survey period. All samples were packed in sealed polythene bags, properly labeled, carried to the laboratory under cooled conditions, and subsequently subjected to serological and molecular detection assays for CMV.

Detection of virus by DAC-ELISA

A survey for the incidence of CMV was conducted in three major chilli-growing districts of Bihar, namely Kishanganj, Purnia, and Bhagalpur, representing two agro-climatic zones. A total of 355 chilli leaf samples showing typical virus-like symptoms were collected during the survey period and initially screened based on field symptomatology (Table 1). Final sample selection for serological testing was made on the basis of symptom severity, frequency of occurrence, and distribution of infected plants within the surveyed fields.

All selected samples were subjected to Direct Antigen Coating Enzyme-Linked Immunosorbent Assay (DAC-ELISA) for CMV detection. The assay revealed that 64 out of 165 samples from Kishanganj, 32 out of 70 samples from Purnia, and 19 out of 95 samples from Bhagalpur reacted positively with CMV antiserum, exhibiting variable absorbance values. Overall, 115 samples tested positive for CMV infection through DAC-ELISA. The district-wise percentage of CMV-positive samples is presented in Table 1.

The data clearly indicate the occurrence of CMV across all surveyed districts, confirming its widespread distribution in chilli-growing regions of Bihar. Positive reactions were determined based on higher optical density (O.D.) values compared with healthy controls, as measured using a microprocessor-based ELISA plate reader. Among all tested samples, the highest O.D. value (0.421) was recorded for an isolate collected from Jalalgarh (Purnia district), which exhibited pronounced shoestring leaf symptoms.

The frequent association of shoestring symptoms with CMV-positive samples observed in the present study is consistent with earlier reports identifying leaf narrowing and filiform as characteristic symptoms of CMV infection in chilli (21–23). These findings validate the usefulness of DAC-ELISA as a reliable method for large-scale screening of CMV under field survey conditions.

Mechanical inoculation and maintenance of virus culture

Mechanical inoculation is a standard method for inducing initial disease symptoms, biological purification, and maintenance of plant viruses in indicator hosts. It involves the transfer of infectious sap from diseased plants to healthy plants through gentle abrasion or minor wounds on the leaf surface without the involvement of insect vectors (24). Even microscopic injuries are sufficient to facilitate virus entry into host cells. Under natural conditions, similar transmission may occur through contact between injured leaves (25).

CMV is readily transmissible through sap inoculation and is known to infect a wide range of host plants, including tomato, chilli, banana, Capsicum annuum, Nicotiana glutinosa, N. rustica, and cucurbits (22, 26). In the present study, mechanical inoculation was employed to biologically purify CMV from infected chilli leaf samples using Chenopodium spp. as an indicator host. Inoculated plants developed mild mosaic symptoms from the sixth day after inoculation, which progressed to severe mosaic symptoms by fifteen days post-inoculation.

For long-term maintenance of virus cultures, hosts with longer life span, ease of cultivation, low susceptibility to sucking pests, and minimal inhibitory compounds in leaf sap are preferred. Based on these characteristics, Nicotiana glutinosa was found suitable for routine maintenance and further experimental use of CMV cultures.

A total of 355 symptomatic samples were initially collected based on visible viral disease symptoms, including mosaic, mottling, chlorosis, leaf deformation, and stunting. All samples were first screened by DAC-ELISA for cucumber mosaic virus detection (Table 2). From these, six representative isolates (Sabour, Kahalgoan, Purnia, Kasba, Belwa, and Kharkhari) were selected for molecular characterization based on: (i) confirmed CMV positivity in DAC-ELISA, (ii) geographic representation across Zone II and Zone IIIB, (iii) Confirmed sap transmission & Maintenance of Virus Culture, iv).variation in symptom expression, and (iv) availability of high-quality RNA suitable for RT-PCR amplification and sequencing of the coat protein gene.

RNA isolation and RT-PCR amplification

Total RNA was extracted from CMV-infected Chenopodium leaves following the protocol described in the Materials and Methods section. The isolated RNA, comprising host genomic RNA along with viral RNA, was used as a template for first-strand cDNA synthesis.

PCR amplification was carried out using CMV coat protein gene-specific primers (Forward: 5′-ATGGTAGTCGTAACAGGTTAG-3′ and Reverse: 5′-TTAGAGGAACTGTTTTAGCCG-3′), along with 10× reaction buffer, dNTPs, and Taq DNA polymerase as described earlier. Thermal cycling conditions were optimized using gradient PCR, and the optimum annealing temperature for amplification of the CMV coat protein gene was determined to be 55 °C for 30 s.

The amplified products were resolved on 1% agarose gel alongside a 1 kb DNA ladder. Successful amplification of the complete coat protein (CP) gene was obtained from six CMV isolates, namely Sabour-chilli, Kahalgoan-chilli, Purnia-chilli, Kasba-chilli, Belwa-chilli, and Kharkhari-chilli, yielding the expected amplicon size of 657 bp (Figure 2).

Analysis of sequencing results

Following sequencing of the cloned CMV coat protein (CP) gene, raw sequence data were quality-checked and edited to remove vector-derived nucleotides and low-quality regions. The cleaned consensus sequences were subsequently submitted to the NCBI GenBank database through the BankIt submission portal. Upon successful validation, accession numbers were assigned to each isolate as mentioned in Table 3.

The accessioned nucleotide sequences obtained in this study were used as reference datasets for comprehensive bioinformatic analyses. All sequences were processed in FASTA format and subjected to multiple sequence alignment, BLASTn-based nucleotide similarity searches, and BLASTp analysis of deduced amino acid sequences against the NCBI database. Each chilli isolate obtained in the present investigation was compared with previously reported Indian and global isolates of CMV to evaluate sequence identity, genetic relatedness, and evolutionary conservation of the coat protein (CP) gene.

Overall, BLASTn analysis revealed a very high level of nucleotide conservation among all six isolates, each with an amplicon length of 657 bp. Sequence identity among the study isolates ranged from 95.8% to 100%, indicating a genetically narrow population structure. Several intra-study comparisons showed complete or near-complete identity (100%), particularly between Kahalgoan and Sabour, Purnia and Kasba, and Belwa and Purnia isolates, suggesting that these isolates are either identical or differ by only single nucleotide substitutions. The limited variation observed was restricted to a few point mutations (single nucleotide polymorphisms), indicating a highly conserved CP gene within the local CMV population.

When compared with previously reported sequences, all isolates showed strong genetic affinity (>96% identity) with Indian CMV isolates such as MN341000.1, MN340995.1, and MN340989.1. In addition, considerable similarity was also observed with geographically distant isolates from Europe, Africa, and other parts of Asia (e.g., AJ810260.1, MN943095.1, KF129062.1), with identity values generally around 97%, indicating strong global conservation of the CP gene. All BLASTn alignments yielded E-values of 0.0 and high bit scores (>1000), confirming statistically significant and reliable homology across comparisons.

At the amino acid level, BLASTp analysis of deduced coat protein sequences further supported the nucleotide findings, revealing a high degree of protein-level conservation with only minor conservative substitutions and no major insertions, deletions, or frame-shift mutations. These substitutions are unlikely to alter the overall structural integrity of the coat protein, suggesting functional stability of the viral CP across isolates.

Collectively, the comparative analyses demonstrate that CMV isolates infecting chilli in Bihar are genetically highly conserved with minimal intra-population diversity. The observed genetic homogeneity, along with close clustering with Indian isolates, suggests a common ancestral origin followed by localized spread and limited evolutionary divergence within the region.

Comparative sequence analysis of present study chilli isolates

To assess coat protein (CP) gene sequences genetic variability among different CMV isolates of the present study such as; Sabour-chilli, Kahalgoan-chilli, Purnia-chilli, Kasba-chilli, Belwa-chilli and Kharkhari-chilli at gene and protein level. All the sequences were aligned using BioEdit software taking as the Purnia chilli as reference sequence. The alignment revealed specific nucleotide substitutions and amino acids at key positions, indicating genomic diversity among the isolates.

At the gene level or nucleotide level, comparative sequence analysis of present study chilli isolates resulted. In the CMV isolates from Sabour and Kahalgoan, a cytosine (C) was observed at position 378, in contrast to the thymine (T) found at the same position in other isolates. This single nucleotide polymorphism (SNP) suggests a possible mutation in the CP gene unique to the isolates from these locations (Figure 3). This nucleotide variation could imply region-specific evolution of the CMV genome, potentially driven by environmental pressures or host interactions.

At the protein or amino acids level Comparative sequence analysis of present study chilli isolates revealed a notable amino acid substitution at position 173. Specifically, valine (V) replaced methionine (M) in the Kahalgoan and Bihar Sharif isolates (Figure 4). This substitution may reflect genetic variability among CMV isolates from different geographical locations.

Phylogenetic analysis

Phylogenetic analysis was performed to elucidate the genetic relationships of the chilli (Capsicum spp.) isolates of CMV obtained in the present study, namely Sabour, Kahalgoan, Purnia, Kasba, Belwa, and Kharkhari, in comparison with representative isolates retrieved from the NCBI GenBank database. These included AY560556 (hot pepper isolate from Thailand), AJ810260 (chilli strain from Germany), D16405 (legume isolate from Japan), D00385 (strain O from Japan), D28780 (NT-6 strain from Taiwan), and Y16926 (tomato isolate from Italy). In addition, CMV isolates from diverse host plants, including banana, jasmine, tobacco, capsicum, chilli, and tomato, were incorporated to enhance the robustness of the phylogenetic framework and to enable a comprehensive assessment of genetic diversity and evolutionary relationships.

The evolutionary history was inferred using the Neighbor-Joining (NJ) method (27). Evolutionary distances were calculated using the Maximum Composite Likelihood model, which provides reliable estimates of nucleotide substitutions among closely related viral isolates. The dataset comprised 30 nucleotide sequences, including six isolates generated in this study and representative reference sequences from different geographical regions.

All positions corresponding to codon sites (first, second, and third) as well as non-coding regions were included in the analysis. Ambiguous positions and gaps were eliminated using pairwise deletion, resulting in a final alignment length of 688 nucleotide positions (28). Phylogenetic reconstruction and all associated analyses were performed using MEGA version 12 (27, 29).

Bootstrap analysis was conducted to assess the robustness of the inferred phylogeny. The resulting phylogenetic tree clearly resolved the clustering patterns among CMV isolates and facilitated the identification of subgroup affiliations, sequence divergence, and probable evolutionary relationships of the Bihar chilli isolates. The placement of the present isolates within established CMV lineages provides important insights into their genetic affinity with previously reported Indian and global isolates. The detailed phylogenetic tree is presented in Figure 5.

The present study provides a comprehensive molecular and epidemiological assessment of CMV infecting chilli (Capsicum annuum L.) in the major production zones (Zone II and Zone IIIB) of Bihar. The integrated approach combining field surveys, serological detection, biological indexing, and RT-PCR-based molecular characterization revealed widespread occurrence of CMV in chilli-growing regions of Kishanganj, Purnia, and Bhagalpur. The disease was associated with characteristic symptoms including mosaic, mottling, chlorosis, vein clearing, leaf deformation, shoestring leaves, puckering, and plant stunting. The variability in symptom severity across locations suggests strong influence of host genotype, environmental conditions, and possible co-infection with other viruses, which is commonly reported in field-grown chilli systems.

Serological confirmation using DAC-ELISA validated the widespread distribution of CMV across both agro-climatic zones, indicating its establishment as a major viral constraint in Bihar chilli ecosystems. Variation in OD values among samples reflects differential viral load and infection stage, consistent with earlier reports demonstrating similar variability in ELISA-based CMV detection (22). These findings reinforce the reliability of serological assays for large-scale epidemiological surveillance.

Biological indexing further confirmed infectivity of representative isolates through successful transmission to indicator hosts (Chenopodium spp. and Nicotiana glutinosa), producing characteristic local lesions followed by systemic infection. The expression of symptoms within 6–15 days post-inoculation confirms the sap-transmissible nature of CMV and aligns with classical experimental evidence (26, 30). These results validate the biological stability and infectivity of CMV isolates circulating in Bihar.

Molecular detection through RT-PCR consistently amplified the expected 657 bp fragment of the coat protein gene from all chilli isolates, confirming the presence of CMV across geographically distinct regions. The high amplification consistency reflects strong conservation of CP gene primer-binding regions and supports the robustness of CP-based molecular diagnostics (30, 31). The uniform detection across all isolates indicates that CMV is genetically stable at the diagnostic target region despite ecological and geographical variation.

Despite overall genetic conservation, minor nucleotide polymorphisms were detected among Bihar isolates, including a substitution at nucleotide position 378 leading to an amino acid change at position 173 in the CP gene. Although limited in number, such substitutions may have biological significance, as CP is a key determinant of host interaction and aphid transmission efficiency.

Previous studies have demonstrated that even single amino acid substitutions in CP can alter symptom expression and host response (32). Similarly, Heo et al. (33) reported extensive genetic variability in CMV populations, particularly within the CP gene, contributing to its broad host range and ecological success. Zečević et al. (34) further highlighted that geographic structuring of CMV populations is often accompanied by specific amino acid changes, reflecting localized evolutionary adaptation.

In this context, the observed amino acid variation in Bihar isolates may represent a region-specific adaptive response driven by host selection pressure and agro-climatic conditions in Eastern India. Although functional validation was not performed in this study, such mutations may contribute to subtle differences in viral fitness, transmission efficiency, or host adaptation.

The present findings indicate that CMV is widely established in chilli production systems of Bihar, with genetically uniform populations belonging predominantly to subgroup IA. The low genetic diversity suggests a stable viral population; however, the presence of minor sequence variation indicates ongoing microevolution. This evolutionary stability may contribute to consistent disease expression across regions, while subtle genetic changes may allow gradual adaptation to local hosts and environments.

The widespread occurrence of CMV across multiple districts highlights the role of alternative hosts, weeds, and aphid vectors in maintaining virus reservoirs. The virus’ broad host range further facilitates its persistence in agro-ecosystems, making eradication impractical and emphasizing the need for integrated management approaches.

The dominance of subgroup IA in Bihar is consistent with earlier Indian studies reporting similar findings in chilli, tomato, and other solanaceous crops (22). Globally, CMV populations show high genetic diversity with frequent recombination and reassortment events; however, CP gene-based studies often reveal conserved clustering patterns due to functional constraints.

Studies from Japan, Taiwan, and Europe have demonstrated that CMV populations exhibit both geographic clustering and host-associated divergence, often linked with amino acid substitutions in the CP region (35). The present study aligns with these observations, indicating that while global CMV populations are highly diverse, regional populations such as those in Bihar may remain genetically constrained within dominant subgroups (Figure 5).

A key outcome of this study is the molecular confirmation that all six representative Bihar isolates (Sabour, Kahalgoan, Purnia, Kasba, Belwa, and Kharkhari) belong to subgroup IA. The clustering of Purnia (OQ756157), Kasba (PV370354), Belwa (PV364319), and Kharkhari (PV364320) was supported by comparatively high bootstrap values, indicating strong statistical confidence and suggesting a recent common evolutionary origin with minimal genetic divergence. Similarly, Sabour (PV364315) and Kahalgoan (PV364316) formed a closely related cluster with high bootstrap support, reflecting stable phylogenetic association and probable localized spread within neighboring chilli-growing regions.

The broader subgroup IA clade containing Bihar isolates together with previously reported Indian and Asian isolates such as AJ810260, MN340989, MN341000, and MN340995 showed moderate bootstrap support, indicating reliable evolutionary grouping while also suggesting gradual diversification among geographically separated CMV populations. According to phylogenetic interpretation standards, bootstrap values above 70% generally represent strong evolutionary reliability, whereas moderate values between 50–70% indicate acceptable but comparatively dynamic evolutionary relationships. The higher bootstrap support observed within Bihar-specific clusters therefore confirms the robustness and reproducibility of local isolate grouping.

In contrast, deeper phylogenetic nodes containing isolates from diverse hosts including banana, jasmine, tomato, and tobacco exhibited relatively lower bootstrap values, suggesting older divergence events and broader evolutionary separation among global CMV populations. Similar observations have been reported by Roossinck (10) and Samad et al. (30), who demonstrated that although the CP gene remains functionally conserved, CMV populations continue to diversify through mutation, host adaptation, and geographic separation.

The strong bootstrap-supported clustering of Bihar isolates indicates low nucleotide variability and high genetic homogeneity among CMV populations infecting chilli in eastern India. These findings further support earlier reports by Ashwathappa et al. (36), which documented the predominance of subgroup IA among Indian CMV isolates. Overall, the bootstrap analysis strengthened the confidence of phylogenetic reconstruction and confirmed that the Bihar isolates represent a stable and evolutionarily related CMV population closely allied with previously reported Indian subgroup IA strains.

The present study provides the first comprehensive molecular characterization of cucumber mosaic virus (CMV) infecting chilli in the major agro-climatic regions of Bihar. The results confirmed the widespread occurrence of CMV and demonstrated that the Bihar isolates predominantly belong to subgroup IA. Phylogenetic and sequence analyses revealed low genetic variability among the isolates, although minor nucleotide and amino acid variations indicated the occurrence of localized microevolution within the virus population. These findings establish an important baseline for understanding the molecular epidemiology and evolutionary dynamics of CMV infecting chilli in eastern India.

The predominance of subgroup IA has significant implications for chilli improvement and disease-management programs in Bihar. Resistance breeding efforts should therefore focus on screening germplasm against subgroup IA isolates to identify resistant or tolerant cultivars suitable for regional cultivation. In addition, integrated disease management strategies involving the use of virus-free seedlings, removal of weed reservoirs, and effective aphid vector management are essential for minimizing disease spread. Early detection through ELISA and RT-PCR-based diagnostics can further support timely disease monitoring and management interventions.

Future studies should focus on characterization of additional genomic regions or complete genome sequencing to obtain deeper insights into recombination events, strain evolution, and subgroup differentiation among CMV populations. Continuous molecular surveillance will also be important for detecting the emergence of new variants and understanding the long-term evolutionary behavior of CMV under changing agro-climatic conditions.

Given the evolutionary potential of CMV, integrating molecular surveillance with breeding and agronomic practices will be critical for sustainable disease management in Bihar chilli production systems.