Abstract
Mango (Mangifera indica), a globally significant fruit crop, is susceptible to diverse pathogens that threaten its productivity. High-throughput sequencing-based virome profiling and Sanger sequencing of symptomatic (mosaic, upward leaf curling with undulated margin in the young leaves) mango samples revealed the presence of a novel olivavirus (mangifera virus 1, MaV-1), and a benyvirus (mangifera indica latent virus, MiLV). Comparative genomic and phylogenetic analyses confirmed the taxonomic placement of MaV-1 within the proposed genus Olivavirus, based on conserved domains and sequence divergence in the RdRp, HSP70h, and coat protein genes. This study represents the first report of MiLV in India and a novel MaV-1 species infecting mango worldwide. Current study enhances understanding of mango viral spectrum and highlights the need for viral surveillance to address the potential threats to mango cultivation.
Supplementary Information
The online version contains supplementary material available at 10.1186/s12985-025-03049-2.
Keywords: Mango, Virome, Benyvirus, Olivavirus, HTS, Phylogenetic analysis, Plant virology, Mango diseases
Introduction
Mango (Mangifera indica), a perennial member of the Anacardiaceae family, is among the most economically and nutritionally significant fruit crops cultivated across tropical and subtropical regions, including India, China, Thailand, Mexico, and Brazil [1–4]. However, mango production is frequently threatened by various biotic stresses, with viral pathogens representing an underexplored but critical constraint to productivity and fruit quality [5, 6]. Virus-infected mango plants may exhibit symptoms such as leaf mosaic, curling and stunting. However, the only previously reported virus (mangifera indica latent virus, MiLV) in mango, is considered latent and has not been associated with pathological symptoms on fruits or leaves [7].
To date, only a limited number of viruses have been reported in mango, including mangifera indica latent virus (MiLV), a benyvirus [7], and mangifera indica totivirus 1 (MitV1), a totivirus [8]. Benyviruses are primarily transmitted through soil-borne plasmodiophorid fungi, such as Polymyxa betae and Polymyxa graminis, which persist in agricultural soils as resting spores and release infectious zoospores under favourable conditions. In addition, mechanical transmission via contaminated irrigation water, soil, or agricultural tools may contribute to their spread, particularly in nursery environments [9]. Such environmental transmission routes raise concerns for inadvertent dissemination through planting material and field operations. On the other hand, viruses in the family Closteroviridae, including olivaviruses, are predominantly transmitted by insect vectors such as aphids, whiteflies, soft scales, and mealybugs in a semi-persistent manner, facilitating virus movement across orchard landscapes.
Conventional diagnostic assays (ELISA, PCR) are limited due to insufficient sequence data for mango viruses. However, the advent of high-throughput sequencing (HTS) technologies due to their ability to detect known and novel viruses without relying on the prior exact sequence information enabled comprehensive analysis of virus communities or virome in plant hosts [10–13]. This has been previously used to reveal characterized as well as uncharacterized virus strains and species [14–16].
In this study, we employed HTS followed by Sanger sequencing-based validation to investigate the virome of symptomatic mango nursery plants displaying mosaic and leaf curling symptoms. The present study aimed to profile the virome of symptomatic mango plants using HTS, validate identified viruses by RT-PCR and Sanger sequencing and characterize their genomes. The study identified a novel closterovirid, mangifera virus 1 (MaV-1), and an Indian isolate of MiLV. In addition to their taxonomic characterization, the potential for environmental and vector-mediated transmission is discussed in the context of mango production systems. The findings underscore the necessity of integrating virome surveillance into phytosanitary protocols for sustainable mango cultivation.
Materials and methods
Sample collection and HTS
Symptoms of mosaic, upward leaf curling with undulated margin of the young leaves were observed in one year old nursery plants of mango (Mangifera indica) cultivar Ambika in Lucknow, India during August 2024. The percent infection of 15–17% was recorded in the nurseries (Fig. 1). The leaf tissues from two symptomatic mango plants (named as MG1 and MG2) were used for RNA extraction. The total RNA was extracted from 100 mg leaf tissues through iRIS method of Ghawana et al. (2011) [17]. The isolated RNA was quantified using a Nano Drop™ (UV–Vis Spectrophotometer, Thermo Fisher Scientific, USA) and assessed for quality on 0.8% agarose gel. The integrity of RNA was quantified through Agilent 4150 TapeStation (Agilent, USA) and Qubit 4 Fluorometer (Invitrogen, USA). Further, the ribozero treatment was given for rRNA depletion using Pan-Plant riboPOOL kit (siTOOLs BIOTECH, Germany) as per manufacturer’s protocol. The libraries were prepared, using the NEBNext Ultra RNA Library Prep Kit for Illumina in accordance with the manufacturer’s instructions (NEB, Ipswich, Massachusetts, USA). Further, libraries were quantified through Agilent 4150 TapeStation (Agilent, USA) and then loaded onto the NovaSeq 6000 (Illumina, USA) platform for paired-end (PE) (2 × 150 bp) sequencing which was outsourced from the commercial facility.
Fig. 1.
Mosaic and leaf curl symptoms in mango (cultivar: Ambika). a Leaves showing mosaic symptoms with mild upward curling, b Leaves showing severe mosaic with pronounced upward curling and undulated margins, especially in young leaves
HTS data pre-processing, identification, annotation and genome analysis
HTS raw reads were imported into the CLC Genomic Workbench (version 20.0.4), where a quality trimming process was applied to eliminate ambiguous and universal adaptor sequences using a Phred quality score cutoff of Q ≥ 30 and a maximum read length of 150 bp. Afterwards, the obtained filtered reads were mapped to the Mangifera indica reference genome (GCA_011075055) obtained from NCBI. Further, the unmapped reads were extracted and de novo assembled with same CLC Genomics Workbench 20 (version 20.0.4) [13]. The obtained contigs were subjected to BLASTx searches using an e-value cutoff of 1e − 5, and only alignments with ≥ 30% identity were retained with existing viral reference genome sequences available at https://www.ncbi.nlm.nih.gov/genome/viruses/ and the NCBI non-redundant database as a reference using the OmicsBox 3.0 (BioBam, Spain). Predictions of conserved domains through viral protein sequences were performed using the NCBI Conserved Domain-Search tool (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) and Pfam database (http://pfam.xfam.org). ClustalW algorithm was used for multiple sequence alignment with classified virus genome sequences and phylogenetic trees were constructed by the best fit maximum-likelihood model using MEGA-X [18]. The analysis incorporated 1000 bootstrap replications to enhance robustness. Additionally, identity matrix was calculated by using Bioedit.
RT-PCR validation and Sanger sequencing
HTS identified mango viruses in the present study were validated through reverse transcription-PCR (RT-PCR). Primer pairs designed from the assembled sequences of identified viruses were used to amplify the genomic regions of identified viruses (Table S1). The first-strand cDNA synthesis was performed with FIRE Script® RT cDNA synthesis kit (Solis BioDyne, Estonia) using 1 μg of total RNA used for HTS of symptomatic samples as well as freshly prepared RNA from one asymptomatic sample, which served as a negative control. The reaction was prepared with 1 µl (5 µM) of each OligodT and random primers, 4 µl of reaction buffer (5X), 1 µl RiboLock RNase Inhibitor (20 U/µl), 2 µl dNTP mix (10 mM) and RevertAid M-MuLVRT 1 µl (200 U/µl). After incubation at 25 °C for 5 min, 42 °C for 60 min, the reaction was stopped by heating at 70 °C for 5 min, and placed on ice. The RT-PCR reaction was performed using 100 ng of cDNA template and 10 μM gene specific primers in an Eppendorf Mastercycler nexus GX2 thermal cycler. The thermocycler amplification, started with one cycle of 94 °C for 4 min, followed by 35 cycles at 94 °C for 30 s, 54 °C to 62 °C for 30 s (the annealing temperature for each virus is given in Table S1), and 72 °C for 1 min, with a final extension at 72 °C for 10 min. The amplified RT-PCR products were then confirmed on 1.6% agarose gel electrophoresis followed by visualization in gel documentation system (BIO-RAD, USA). Furthermore, the cloning of amplified RT-PCR product was performed followed by Sanger sequencing to confirm the sequences of amplified PCR products.
Results and discussion
Approximately 43 million raw reads were obtained from the RNA of both the mango leaf samples (MG1 and MG2). The quality trimmed clean reads were aligned to the mango genome sequences and unaligned reads were then considered for the virus identification. The unaligned reads of mango virome were found in the range of 3,144,006 to 3,383,175, representing 7.39-7.71% of the total reads. De novo assembly of unaligned reads with CLC Genomic Workbench (version 20.0.4) yielded 522 and 370 contigs longer than 200 bp in MG1 and MG2, respectively. The N50 value of contig length was in a range of 1,511 to 1,508 in MG1 and MG2, respectively (Table 1). The homology analysis for virus identification using BLASTx algorithm against NCBI non-redundant database resulted assembled contigs mapped with a known benyvirus (mangifera indica latent virus) and a novel olivavirus belonging to viral families, Benyviridae and Closteroviridae, respectively. The MG1 and MG2 revealed the five contigs ranging from 4,182 nt to 17,243 nt that showed 34.03–56.52% nt sequence identity with Olivavirus and in case of MiLV, the BLASTx analysis of seven contigs ranging from 6741 to 2518 nt showed 97.66–95.45% nt sequence identity to RNAs 1 and 2 of MiLV isolates in GenBank. The complete genomic RNA sequences of olivavirus and benyvirus were assembled from MG1 and MG2, respectively.
Table 1.
Summary statistic of two mango transcriptome
| Samples | Statistics terms | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Total raw reads | Total filtered reads (HQ) | Host aligned reads | Host unaligned reads | De novo assembly | MaV-1 | MiLV | ||||
| N50 | Contig length | Map read | Genome coverage (X) | Map read | Genome coverage (X) | |||||
| MG1 | 40,778,152 | 40,771,310 | 37,633,224 | 3,144,006 | 1,511 | 17,243–304 |
110,096 (3.96%) |
453 |
670 (0.02%) |
297 |
| MG2 | 45,781,178 | 45,772,166 | 42,398,003 | 3,383,175 | 1,508 | 8,398-207 |
10,379 (0.5%) |
67 |
9,310 (0.32%) |
174 |
Genome characterization of novel virus
The homology analysis (BLASTx algorithm) revealed that the contig of 17,243 nt (Sample: MG1) shared identity to actinidia virus 1 (genus: Olivavirus) (Accession No: OK513319) (34.03% aa identity; 31% query coverage; E-value: 0.0). The contig was predicted to have ten ORFs encoding proteins involved in replication, structural assembly and putative host-virus interactions (Fig. 2a). The polyprotein ORF1a (6,474 nt; 2,158 aa), with a molecular weight (MW) of 237.4 kDa encodes two proteins: a helicase (HEL, pfam01443) and a methyltransferase (MET, pfam05417). ORF1b (1,653 nt; 551 aa), with a MW of 60.6 kDa, encodes a putative RdRp (pfam00680), which is crucial for viral RNA synthesis. ORF2, 3, 6, 8 and 9 (17.2–37.4 kDa; 468–1,020 nt; 156–340 aa) encodes several putative hypothetical proteins. Their functions remain uncharacterized but may contribute to viral replication and host adaptation. ORF4 (64.7 kDa; 1,764 nt; 588 aa) encodes a putative HSP70 (pfam00012) and ORF5 (54.7 kDa; 1,491 nt; 497 aa) encodes a putative HSP90 (pfam03225), suggesting its role in protein stabilization during viral infection and in host stress response modulation. ORF7 (25.4 kDa; 693 nt; 231 aa) encodes a putative CP (pfam01785).
Fig. 2.
Schematic presentation of genome organization: a mangifera virus 1 and b mangifera indica latent virus
Comparison of the amino acid sequences of three taxonomically important proteins, RdRp, HSP70h, and CP with homologous proteins from Closteroviridae family which included ampeloviruses, olivaviruses and velariviruses revealed sequence identities ranging from 27.4% to 47.1%, 29.8 to 39.9 and 20.5% to 32.6% (Table 2). Among olivaviruses, the three proteins showed highest aa sequence identity with actinidia virus 1 (KX857665), as 47.1% in RdRp, 39.9% in HSP70h and 32.6% in CP (Table 2). The species demarcation criteria for the unclassified or yet to be classified Olivavirus genus (family: Closteroviridae) are typically defined as having less than 75% amino acid identity in key gene products (RdRP, CP and HSP70h) [19]. Based on ICTV species demarcation criteria, the viral genome sequence annotated in this study was attributed to a new Olivavirus species, named as mangifera virus 1 (MaV-1). Phylogenetic analysis of the RdRp, HSP70h, and CP sequences from closteroviruses was conducted using the best fit maximum-likelihood method in MEGA-X. The best-fit substitution models selected were LG + G + I for RdRp, LG + G + I + F for HSP70h and LG + G + I for CP with 1,000 bootstrap replicates. The RdRp, HSP70h and CP sequence of MaV-1 was clustered with olivaviruses (Fig. 3a). This supports the classification of MaV-1 as a new species within the genus Olivavirus. The complete genomic RNA sequence (17,243 nt) of the MaV-1 was submitted to GenBank with accession number PV101221. As the 5′ and 3′ termini were not validated by RACE, this sequence should be considered near-complete genome/complete coding genome sequence.
Table 2.
Sequence identity of pairwise combinations of RdRp, HSP70 and CP sequences of mangifera virus 1 (PV101221) with other sequences of olivaviruses, classified velariviruses and ampeloviruses
| Virus | Accession No |
% aa identity | |||
|---|---|---|---|---|---|
| Genus | Species | RdRp | HSP70 | CP | |
| Olivavirus | Actinidia virus 1 | KX857665 | 47.1 | 39.9 | 32.6 |
| Persimmon virus b | AB923924 | 36.3 | 31.2 | 20.5 | |
| Olive leaf yellowing-associated virus | AJ440010 | 27.4 | 29.8 | - | |
| Velarivirus | Cordyline virus 1 | HM588723 | 21.8 | 23.1 | 07.1 |
| Little cherry virus 1 | EU715989 | 04.9 | 20.5 | 05.6 | |
| Grapevine leafroll-associated virus 7 | HE588185 | 23.0 | 22.3 | 07.2 | |
| Ampelovirus | Grapevine leafroll-associated virus 1 | NC016509 | 22.5 | 19.6 | 07.8 |
| Grapevine leafroll-associated virus 3 | NC004667 | 05.2 | 20.7 | 05.6 | |
| Little cherry virus 2 | NC005065 | 06.3 | 19.7 | 06.0 | |
Fig. 3.
Phylogenetic analysis of a mangifera virus 1 and b mangifera indica latent virus. Phylogenetic tree was generated using the best fit maximum-likelihood model using MEGA-X 1,000 bootstrap replicates (places at the nodes on the tree)
Genome characterization of mangifera indica latent virus
In case of MiLV, the BLASTx analysis of contigs 6741 nt and 2518 nt (Sample: MG2) showed 97.66 (91% query coverage; E-value: 0.0) and 98.45% (42% query coverage; E-value: 0.0) aa sequence identity to RNA 1 (KU140662) and RNA 2 (KU140663) of MiLV isolate in GenBank, respectively. A total of 3,26,152 reads were mapped to MiLV RNA 1 and 2. The complete genomic RNA1 (6741 nt) and RNA2 (2518 nt) sequences of the Indian MiLV was determined and submitted to GenBank with accession numbers PV199041 and PV199042. It shared the same genome organization as that of previously reported Israel isolate [7]. The RNA1 is 6741 nt, encoding RNA-dependent RNA polymerase of 624 aa from 127 to 6285 nt. This contains the viral (Superfamily 1) RNA helicase (PF01443) and RNA-dependent RNA polymerase domain (PF00978). The RNA2 is 2518 nt with four ORFs, ORF1 encoding the putative coat protein (PF00721) of 182 aa from 119 to 667 nt. ORF2 encodes the putative viral helicase protein (PF01443) of 249 aa from 755 to 1813 nt. ORF3 encodes the putative movement protein (PF01307) of 98 aa from 1782 to 2168 nt and ORF4 encodes putative hypothetical protein of 97 aa from 2110 to 2463 nt (Fig. 2b).
Comparison of the amino acid sequences of two taxonomically important proteins RdRp and CP from MiLV with homologous proteins from other classified benyviruses, revealed sequence identities ranging from 36.8% to 97.6% and 12.8% to 99.4% with classified Benyvirus species (Table 3). Present isolate of MiLV shared highest identity with previously reported MiLV from Israel (KU140662). Phylogenetic analysis of the RdRp and CP sequences from benyviruses, conducted using the best fit maximum-likelihood model, showed that RdRp and CP sequence of MiLV clustered with Israel isolate of MiLV (Fig. 3b). Currently, only one complete genomic RNA1 and RNA2 sequences of MiLV isolate from Israel is available in public domain [7]. Present study reports the second near-complete genome/complete coding genome sequence of MiLV and the first from India. In RT-PCR based validation, out of three samples, both HTS samples were positive for MaV-1 and MiLV (Fig. S1). The amplified products were Sanger sequenced, obtained sequences were assembled and submitted to NCBI GenBank.
Table 3.
Sequence identity of pairwise combinations of RdRp and CP sequences of mangifera indica latent virus (PV199041 and PV199042) with other sequences of classified benyviruses
In conclusion, the discovery of new viruses in mango not only enhances our understanding on virus diversity, it also provides information on the phytosanitary status of plants which is important for perennial crops in order to produce certified healthy planting material. The application of HTS for virome study highlights the necessity for its use in virus diagnostics to improve the virus indexing programme in fruit trees.
Supplementary Information
Acknowledgements
The authors are thankful to the Joint Director (Research) and Director, ICAR-IARI, New Delhi for providing facilities to carry out the study.
Author contributions
M.R.P.: Formal analysis, investiga¬tion, methodology, writing—original draft preparation. M.Y.S.: Validation., J.G.: Validation., A.T.: Validation. D.S.: Validation. N.G.: Writing—review and editing. S.K.S.: Conceptualization, supervision, resources, methodology, writing—review and editing. V.K.B.: Conceptualization, methodology, supervision, writing—review and editing.
Funding
The research work was supported by the lndian Council of Agricultural Research, Department of Agricultural Research and Education, Government of lndia under National Professor Scheme (Ag.Edn.F.No./27/01/NP/2022-HRD) and All India Network Project on Emerging Pests (AINP-EP)-Plant Virus Sub-component (CS 8–13/2024-PP).
Data availability
The raw datasets supporting this study will be accessible upon publication in the Sequence Read Archive (SRA) repository with accession numbers: SRR32540434 and SRR32540435. The genomic RNA sequence of viruses have been deposited in NCBI under accession number PV101221, PV199041 and PV199042.
Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Malyaj R. Prajapati, Mailem Yazing Shimray and Jayesh Gehlot contributed equally to this work.
Contributor Information
Nitika Gupta, Email: nitika.iari@gmail.com.
Virendra K. Baranwal, Email: vbaranwal2001@yahoo.com
Susheel Kumar Sharma, Email: susheelsharma19@gmail.com.
References
- 1.Tharanathan R, Yashoda H, Prabha T. Mango (Mangifera indica L.), “the king of fruits”—an overview. Food Rev Int. 2006;22:123–95. 10.1080/87559120600574493. [Google Scholar]
- 2.Bura S, Jasrotia A, Sharma S, Sharma A, Tutlani A. Recent advances in breeding of mango (Mangifera indica): a review. Int J Environ Clim Change. 2023. 10.9734/ijecc/2023/v13i113196. [Google Scholar]
- 3.Mitra S. Mango production in the world – present situation and future prospect. Acta Hortic. 2016;1111:287–96. 10.17660/ACTAHORTIC.2016.1111.41. [Google Scholar]
- 4.Priya S, Singh H. Variability in area, production, productivity and export of mango. Int J Curr Microbiol Appl Sci. 2020;9:1409–13. 10.20546/ijcmas.2020.902.163. [Google Scholar]
- 5.Subramaniam S, Noordin N, Azrizal-Wahid N, Zainudin N. Insects induced mango fruit rot: unraveling the culprits. Andalasian Int J Entomol. 2024. 10.25077/aijent.2.1.48-59.2024. [Google Scholar]
- 6.Jiang T, Zhou T. Unraveling the mechanisms of virus-induced symptom development in plants. Plants. 2023. 10.3390/plants12152830. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Sela N, Luria N, Yaari M, Prusky D, Dombrovsky A. Genome sequence of a potential new benyvirus isolated from mango RNA-seq data. Genome Announc. 2016;4(6):10–1128. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Chen M, Xia Y, Wang Q. Identification and molecular characterization of a novel totivirus from Mangifera indica. Arch Virol. 2024;169(3):58. [DOI] [PubMed] [Google Scholar]
- 9.Lee G, Lee S, Kim M, Jeong R. Aquatic environments as vectors for plant virus spread: challenges and solutions. Res Plant Dis. 2025. 10.5423/rpd.2025.31.1.30. [Google Scholar]
- 10.Villamor D, Hồ T, Rwahnih M, Martin R, Tzanetakis I. High throughput sequencing for plant virus detection and discovery. Phytopathology. 2019;109(5):716–25. 10.1094/PHYTO-07-18-0257-RVW. [DOI] [PubMed] [Google Scholar]
- 11.Sidharthan VK, Baranwal VK. Public domain databases: a gold mine for identification and genome reconstruction of plant viruses and viroids. In: Genomics data analysis for crop improvement. Singapore: Springer Nature; 2024. p. 247–84.
- 12.Prajapati MR, Manav A, Singh J, Kumar P, Kumar A, Kumar R, et al. Identification and characterization of a garlic virus E genome in garlic (Allium sativum L.) using high-throughput sequencing from India. Plants. 2022;11(2):224. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Prajapati MR, Singh J, Kumar P, Kumar R, Baranwal VK. Identification of a novel member of the Alphaendornavirus genus in Plantago ovata through transcriptome dataset mining. S Afr J Bot. 2023;157:135–7. [Google Scholar]
- 14.Bejerman N, Dietzgen R, Debat H. Novel tri-segmented rhabdoviruses: a data mining expedition unveils the cryptic diversity of cytorhabdoviruses. Viruses. 2023;15(12):2402. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Prajapati MR, Diksha D, Thapa P, Sharma SK, Gupta N, Baranwal VK. Identification of a novel mitovirus in grapevine through high-throughput sequencing. Int Microbiol. 2024. 10.1007/s10123-024-00572-0. [DOI] [PubMed] [Google Scholar]
- 16.Prajapati MR, Gupta N, Thapa P, Diksha D, Sharma SK, Baranwal VK. Identification and characterization of a novel cytorhabdovirus associated with pear (P. communis ‘Bartlett’) through RNA-seq. Curr Microbiol. 2025;82(1):39. [DOI] [PubMed] [Google Scholar]
- 17.Ghawana S, Paul A, Kumar H, Kumar A, Singh H, Bhardwaj PK, et al. An RNA isolation system for plant tissues rich in secondary metabolites. BMC Res Notes. 2011;4:1–5. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018;35(6):1547–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Blouin AG, Biccheri R, Khalifa ME, Pearson MN, Poggi Pollini C, Hamiaux C, et al. Characterization of Actinidia virus 1, a new member of the family Closteroviridae encoding a thaumatin-like protein. Arch Virol. 2018;163:229–34. [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The raw datasets supporting this study will be accessible upon publication in the Sequence Read Archive (SRA) repository with accession numbers: SRR32540434 and SRR32540435. The genomic RNA sequence of viruses have been deposited in NCBI under accession number PV101221, PV199041 and PV199042.