Molecular characterization and genetic diversity of papaya meleira virus 2 associated with papaya sticky disease in northeast BrazilABSTRACT
Papaya sticky disease is one of the main diseases of agricultural crops in Brazil. It is characterized by the spontaneous exudation of latex in the fruits, which oxidizes, giving the fruit a sticky appearance. These symptoms are associated with a mixed infection formed by the papaya meleira virus and the newly discovered papaya meleira virus 2 (PMeV2). This work aimed to molecularly characterize the first PMeV2 isolate from northeastern Brazil; to estimate the phylogenetic relationships of this isolate with the other viruses of the Tombusviridae family; and in addition, to estimate the genetic diversity and phylogenetic relationships between PMeV2 isolates. The obtained sequence was named PMeV2-RN, which has 4,435 nucleotides and showed 94% identity with the isolate PMeV2-ES from Espírito Santo, Brazil. The sequence contains two predicted open reading frames (ORFs) in different reading phases. ORF1 encodes a 238 amino acid polypeptide that has 88% identity with the corresponding PMeV2-ES protein. ORF2 encodes a protein of 473 amino acids that presents 100% identity with the RNA-dependent RNA polymerase (RdRp) protein of PMeV2-ES. Phylogenetic analyses showed a greater proximity between isolate PMeV2-ES and isolates papaya virus Q (PpVQ) and PMeV-Mx than with PMeV2-RN, which seems to belong to a distinct lineage of this virus in Brazil. The comparative analysis of the nucleotide sequences among the isolates obtained in different producing regions of the northeast region of Brazil indicated a high degree of similarity between the sequences. It showed a high degree of conservation among the isolates, which did not form geographically structured groups.
Key words
Papaya sticky disease; PMeV; papaya viruses;
Umbravirus
; Totiviridae
INTRODUCTION
Brazil is one of the largest papaya exporters in the world, with its cultivation concentrated in the northeast region, covering three of the four main papaya-producing states in the country: Bahia, Ceará, and Rio Grande do Norte (IBGE 2019). Despite Brazil’s prominent position, the papaya crop productivity and economic performance have suffered due to phytosanitary problems, especially because of the papaya sticky disease. The disease, caused by the papaya meleira virus (PMeV), is one of the main viral diseases that affect papaya crops in the country (Abreu et al. 2015). It was first reported in Brazil in the early 1980s and later in Mexico in 2008 (Kitajima et al. 1993, Perez-Brito et al. 2012).
Papaya sticky disease is characterized by a spontaneous exudation of latex in the fruits, which oxidizes and gives it a sticky appearance. In some cases, the flavor of the pulp may be affected. Necrotic symptoms may appear on the edges of young leaves because of the latex exudation (Abreu et al. 2015). The etiologic agent of the disease is the PMeV, characterized by having an isometric particle and a genome composed of a double-stranded RNA molecule of approximately 8.8 kb (Abreu et al. 2015). Analyses of the genetic diversity of PMeV using a fragment of approximately 560 bp of RNA-dependent RNA polymerase (RdRp) indicated similarities with mycoviruses of the Totiviridae family (Daltro et al. 2014). The ORF2 of PMeV has characteristic conserved domains of RdRps present in mycoviruses of the Totiviridae family (Abreu et al. 2015).
Studies carried out in Brazil demonstrated the presence of a new virus associated with papaya plants that showed symptoms of papaya sticky disease. In RNA samples extracted from PMeV infected papaya latex, a fragment of approximately 4.5 kb was observed, which was first attributed to the presence of a subgenomic RNA (Maciel‐Zambolim et al. 2003). Later, it was observed that it was a single-stranded virus (ssRNA), with approximately 4.5 kb, which was named papaya meleira virus 2 (PMeV2) (Antunes et al. 2016). This second virus has no genetic relationship with PMeV, but it is related to PMeV-Mx, identified in Mexico, and papaya virus Q (PpVQ), identified in Ecuador, both closely related to the genus Umbravirus (Perez-Brito et al. 2012, Quito-Ávila et al. 2015).
Commonly, viruses of the genus Umbravirus are dependent on other viruses for the encapsulation of their RNA. In view of this, studies conducted by Antunes et al. (2016) indicated that PMeV appears to act as a helper virus for PMeV2. The PMeV protein coat forms hybrid virus particles encapsidating the ssRNA of PMeV2. Preliminary studies indicate that the induction of typical symptoms of the disease is associated with double infection of PMeV and PMeV2 (Antunes et al. 2016).
The characterization of a new virus associated with papaya sticky disease is essential for a better understanding of its pathosystem, as well as for the relationship between plants and viral complexes. The development of more knowledge can subsidize the establishment of new diagnoses and more efficient ways to control the disease. In this context, the present study aimed to carry out the molecular characterization of a PMeV2 isolate; to estimate the phylogenetic relationships of this isolate with other viruses of the Tombusviridae family, and, based on sequencing of the genomic region of the RdRp, to estimate the genetic diversity and phylogenetic relationships between PMeV2 isolates from papaya-producing regions in northeastern Brazil.
MATERIAL AND METHODS
Selection of isolates from northeastern Brazil
We included in this study sequences of seven isolates from Bahia, nine isolates from Rio Grande do Norte, and one from Espírito Santo (Table 1).
List of papaya meleira virus 2 (PMeV2) isolates studied during the development of this work and their respective locations.
Sequencing and genomic analysis
The latex sample used for total RNA extraction was collected in 2009 from a papaya tree that showed well-known symptoms of papaya sticky disease. Plants were in a cultivation located in the municipality of Baraúnas, state of Rio Grande do Norte. The sample yielded PMeV2 isolate, designated as PMeV2-RN. The entire process of obtaining the RNA, sequencing, and assembling the contigs was previously performed by Abreu et al. (2015), and it is described below.
Total RNA was extracted using Trizol (Thermo Fisher Scientific) following the manufacturer’s instructions and then stored in a freezer at -20°C. Sequencing of a 30 µg RNA sample was performed at Macrogen Inc. (Seoul, South Korea). Such RNA samples were used to obtain cDNA libraries from random hexamers. The cDNA libraries were sequenced on a 454 GS-FLX Titanium platform. The contigs were assembled using the Geneious 5.4.5 software (Drummond et al. 2011). The sequences were compared to those deposited in the GeneBank database using the BLASTx tool, which indicated the sequences most similar to those obtained in this study. Using Geneious 5.4.5, the contigs that showed homology with viruses were chosen and used for editing and assembly of larger contigs. The ORF Finder tool was used to detect open reading frames (ORFs) that corresponded to known viral genes. Most similar virus genomes to PMeV2-RN sequences were used to determine their structure and genomic organization, as well as to define their taxonomic classification.
PMeV2-RN sequence analysis and phylogenetic relationships between PMeV2-RN and species of the family Tombusviridae
The genomic sequence of the PMeV2-RN isolate was compared with the genome of 17 other species belonging to the Tombusviridae family (Table 2). Phylogenetic analyses were performed using the principles of maximum likelihood (ML) and Bayesian inference (BI). ML analysis was performed using MEGA X software (Kumar et al. 2018) with the Hasegawa-Kishino-Yano (HKY) substitution model and a bootstrap of 1,000 repetitions to assess the reliability of each clade. BI analyses were conducted using the HKY+G model. Hierarchical likelihood tests based on the Akaike information criterion were used, being carried out in the jModelTest2 program (Darriba et al. 2012) and implemented on the CIPRES Science Gateway platform (Miller et al. 2010) . The analysis was carried out in MrBayes 3.2.2 software (Ronquist and Huelsenbeck 2003), conducted in the CIPRES Science Gateway platform (Miller et al. 2010)1. Two runs of four Markov chain Monte Carlo chains each were executed for 5,000,000 generations. The analysis was carried out in MrBayes 3.2.2 software (Ronquist and Huelsenbeck 2003), conducted in the CIPRES Science Gateway platform (Miller et al. 2010), with two independent runs with four chains (two hot and two cold) executed for 5,000,000 generations. One tree was sampled every 1,000 generations, 25% of initial trees were discarded as burn-in. The posterior probability (PP) of each clade was estimated. The tree was visualized and edited in the FigTree 1.4.4. Bootstrap support (BS) and PP values lower than 75% were not mentioned.
Name, abbreviation, accession numbers, genetic identity percentage of viral sequence with the sequence of the PMeV2-RN isolate, and genus of the viruses of the family Tombusviridae used in the phylogenetic analysis.
Study of genetic diversity and phylogenetic relationships among PMeV2 isolates from northeastern Brazil
The genetic identity between the sequences was accessed with the aid of the Clustal W Program (Thompson et al. 1994). To analyze the genetic diversity among the isolates, a genetic distance matrix based on the p-distance substitution model (Nei and Kumar 2000) was generated and used for cluster analysis with the Neighbor-Joining algorithm using the MEGA X software (Kumar et al. 2018). The phylogenetic analysis was performed using the principle of ML, and the evolutionary model adopted was HKY+G using MEGA X. Sequences from PpVQ (Ecuador) (KP165407.1) and PMeV-Mx (Mexico) (KF214786) isolates were used as outgroup.
RESULTS
PMeV2-RN sequencing and genomic analysis
Sequencing of PMeV2-RN isolate generated 96,215 readings, with an average length of 400 nucleotides (nt), totaling 39,081,589 nt. After editing the fragments, 519 contigs were obtained, among which a 4,435-nt contig with high homology with the PMeV2-ES (98%) previously identified in Espírito Santo in 2016 (Antunes et al. 2016). The sequence generated in this work was named PMeV-RN, referring to the state where the latex sample was collected (Table 1).
PMeV2-RN genomic sequence also shares 72 and 70% identity with PMeV-Mx and PpVQ, respectively, both related to members of the genus Umbravirus (Quito-Ávila et al. 2015, Zamudio-Moreno et al. 2015).
Sequence analysis allowed the identification of two predicted ORFs in different reading phases (Fig. 1). ORF1 (nt 717-3614) encodes a 238 amino acid (aa) polypeptide that has 88% identity with the corresponding hypothetical PMeV2-ES protein and 47.5% identity with the PMeV-Mx protein. ORF2 (nt 2031-3452) codes for a 473 aa protein and showed 100% identity with the RdRp protein of PMeV2-ES. The ORF2 product presented, respectively, 73 and 66% identity with the RdRps of PpVQ and PMeV-Mx, as well as 44% identity with the RdRps of ethiopia maize-associated virus (AWS06679.1) and opuntia umbra-like virus (AXG65483), 42% identity with the RdRp of citrus yellow vein-associated virus (CYVaV; YP_009551334) and 42% identity with the RdRp of carrot mottle virus (CMoV) (BBH43057.1).
Schematic representation of papaya meleira virus (PMeV2-RN) genomic organization. The viral single-stranded virus has two open reading frames (ORFs) in different reading phases. ORF1 encodes a hypothetical protein, and ORF2 encodes a putative RdRp.
Phylogenetic relationships between PMeV2-RN and viruses of the family Tombusviridae
Using ML and BI methods, the phylogenetic analyses, which were performed to infer the evolutionary relationships between PMeV2-RN, PpVQ, PMeV-Mx, and 17 species of the family Tombusviridae (Table 2), produced trees with similar topologies, especially in relation to the most supported clades. This indicates that the phylogenetic relationships evidenced in this work were not influenced by the phylogenetic analysis methods employed, and, for this reason, it was decided to present and discuss only the tree with highest likelihood (Fig. 2).
The tree with the highest likelihood obtained from the nucleotide sequences of papaya meleira virus 2 (PMeV2)-RN and species of the Tombusviridae family. It shows the phylogenetic relationships between PMeV2-RN and related viruses. Bootstrap (in black) and posterior probability (in red) values are above branches. The virus names used in the analysis and their respective Genbank accession numbers are listed in Table 2. The scale bar represents the number of substitutions per site.
The tree with highest likelihood had high support (greater than 75%) of boostrap and PP for clades formed among isolates PMeV2-ES, PpVQ, PMeV-Mx (Clade 1); TCV, GaMV and OLV-1 (Clade 2); OMMV and TNV-A (belonging to the genus Alphanecrovirus) (Clade 4); MNSV, groundnut rosette umbravirus (GRV) and OpPMV (Clade 5). Clades 1 and 2 grouped together with CMoV and TBTV isolates forming clade 3.
Phenetic and phylogenetic analyses among PMeV2 isolates
The genomic region chosen for the sequence analysis corresponds to the gene that encodes the RdRp of 473 aa. Comparative analysis showed high degree of identity among the isolate sequences, between 85.9 and 99.8% (Table 3). When compared to the RdRp nucleotide sequence of PpVQ and PMeV-Mx, which belong to the genus Umbravirus, the levels of identity varied between 67.3–71.3 and 64.8–67.2%, respectively (Table 3).
Genetic identity matrix (%) derived from RNA-dependent RNA polymerase (RdRp) nucleotide sequences (isolates from northeastern Brazil) and papaya meleira virus (PMeV)-ES, PMeV-Mx and papaya virus Q (PpVQ).
Cluster analysis revealed that the isolates collected in Brazil form a large highly supported group (BS 100%), except for the isolate PMeV2-RN (Fig. 3a). The isolates from Rio Grande do Norte, except for isolate PMeV2-RN, formed a group with 90% BS (group 1). However, this group also includes isolate 216, from Bahia. The other isolates from Bahia created two distinct groups, a small group, formed by isolates 211, 215, and 240 (group 2, BS 80%), and a second one including the remaining isolates from Bahia and all isolates from Rio Grande do Norte (group 3, BS 95%). The only exception was isolate 04, which appeared as the most distant from the others, occurring at the base of the grouping of most Brazilian isolates, after the isolate from Espírito Santo (BS 100%). Additionally, isolate from Espírito Santo, as well as isolate 04, from Bahia, showed great genetic divergence in relation to the others, not grouping with any other isolate in this study (Fig. 3a).
Genetic diversity and phylogenetic relationships among PMeV2 isolates from northeastern Brazil: (a) dendrogram of isolates from northeastern Brazil based on the replicase gene (PMeV2 RdRp), using p-distance substitution model and Neighbor-Joining algorithm. Numbers above branches indicate bootstrap values (10001,000 repetitions). (b) The tree with the highest likelihood obtained from the analysis of the genomic region of the replicase gene (PMeV2 RdRp). The isolates were obtained from the latex of papaya trees, located in northeastern Brazil, with symptoms of papaya sticky disease. The numbers presented on the branches indicate bootstrap support with 10001,000 repetitions (–(-ln = 0.597015).
Phylogenetic analysis carried out using ML analysis (Fig. 3b) showed that the Brazilian isolates, except for PMeV2-RN, form a single highly supported monophyletic group (BS 100%), although internal clades are not well supported, with the exception of small clades formed by two or three isolates from the same state. Isolate PMeV2-ES, from Espírito Santo, and isolate 04, from Bahia, also formed a small supported clade (BS 93%).
DISCUSSION
Genomic analysis of PMeV2-RN
This is the first study to confirm the presence of PMeV2 in the northeastern region of Brazil, an important papaya production region of the country. The contig of 4,435 nt share high homology (94%) with PMeV2-ES isolate, so we referred this isolate as PMeV2-RN, and potentially represents a new species of the genus Umbravirus. PMeV2-RN also showed high similarity with PpVQ from Ecuador (Quito-Ávila et al. 2015) and the isolate PMeV-Mx, detected in papaya plants with similar symptoms to papaya sticky disease in orchards in Mexico (Perez-Brito et al. 2012). The genetic identity percentages between the sequence of PMeV2-RN and the available sequences of PMeV-Mx and PpVQ (72 and 70%, respectively) were similar to those detected between PMeV2-ES and the same isolates (79 and 70%, respectively) (Antunes et al. 2016). These values are close to the limit of 70% determined for the demarcation of species in the genus Umbravirus (Ryabov et al. 2012). However, as the PMeV-Mx and PpVQ genomes have not been completely sequenced yet, it is not possible to determine whether these viruses are distinct isolates of the same species or different species. Furthermore, biological and genomic differences observed among these viruses justify further analysis to determine whether these viruses should be included in the Umbravirus genus or belong to a distinct genus of umbra-related viruses.
Commonly, Umbraviruses have genomes ranging from 4 to 4.5 kb containing up to four ORFs (Simon et al. 2024). They differ from most other plant-infecting viruses as they do not encode a conventional capsid protein and rely on the presence of a helper virus — usually from the Luteovirus, Enamovirus, Polerovirus, and Sobemovirus genera — for RNA encapsidation, allowing their plant-to-plant transmission by insect vectors. In turn, Umbraviruses provides a cell-to-cell MP that mitigates the normal phloem restriction of the helper virus (Taliansky and Robinson 2003). The PMeV-2 genome contains two non-overlapping ORFs (ORF1 and ORF2), followed by an 1,800-nucleotide-long non-coding region. Besides the absence of a canonical MP identified on the PMeV2-RN genome, it is believed that the protein encoded by ORF1 could be involved in the movement of PMeV in the plant. Analysis of the protein encoded by ORF2 revealed a conserved domain of the RdRp superfamily 3 (pfam 00998: RdRP 3), which includes the RdRp of several plant viruses. The amino acid sequence shares high identity with PMeV-2-ES (98.4%), and with PpVQ and PMeV-Mx (70% each) (data not shown).
In summary, as there is no evidence of the presence of any virus from the genera commonly associated with Umbraviruses in papaya plants with sticky symptoms, we believe that in the papaya sticky disease complex, PMeV acts as a helper virus to PMeV2. This hypothesis is supported by mass spectrometry sequencing of purified PMeV virions, which confirmed the presence of PMeV2 RNA (Antunes et al. 2016).
Phylogenetic relationships between PMeV2-RN and viruses of the family Tombusviridae
Phylogenetic analyses of isolates of the Tombusviridae family revealed that PMeV2-ES, PpVQ, and PMeV-Mx isolates form a highly supported clade (Fig. 2) (Clade 1).
The isolate PMeV2-RN, even showing great genetic similarity with those isolates, was distantly related to Clade 1, constituting a sister group, although without support, of a large group that includes the PMeV2-ES, PpVQ, and PMeV-Mx isolates and others of the Tombusviridae family. These results reinforce our hypothesis that PMeV2-ES, PpVQ, and PMeV-Mx isolates may belong to the same Umbravirus group or to another virus closely related to this genus. Also indicates that PMeV2-RN and PMeV2-ES isolates constitute distinct strains of this virus in the Brazilian territory or represent distinct infection events.
Phenetic and phylogenetic analyses among PMeV2 isolates
The isolates collected in the northeast region showed a high degree of genetic identity between their RdRp nucleotide sequences (85.9 to 99.8%), demonstrating a high degree of conservation, even among those from distant regions and collection intervals (Table 3). For instance, isolate 152, from Rio Grande do Norte, even having been collected eight years before the other isolates, also showed a high level of identity (88.8 to 98.7%) with the other isolates.
Similar results were also observed in studies with ssRNA viruses. High values of identity were found among isolates collected from distant regions, such as: potato virus M (PVM), cherry virus A (CVA), blueberry scorch virus (BlScV), grapevine fanleaf virus (GFLV), and cherry leaf roll virus (Ge et al. 2014, Woo and Pearson 2014, Zhou et al. 2015, Gao et al. 2017). Studies on the genetic diversity of the capsid protein of the viral complex that causes groundnut rosette disease, which includes an Umbravirus (GRV), in the African region, observed that the capsid protein gene was highly conserved (97 to 99%), regardless of its geographic origin (Deom et al. 2000).
Cluster analysis demonstrated that the Brazilian isolates form a highly supported group (BS 100%), except for isolate PMeV2-RN, which differed significantly from the others (Fig. 3a). The isolates from Rio Grande do Norte, apart from PMeV2-RN isolate, formed a group with 90% BS (group 1), but this group also includes isolate 216, from Bahia. The other isolates from Bahia were grouped into two distinct groups. A small group was formed by isolates 211, 215, and 240 (group 2, BS 80%), and another with the other isolates from Bahia, close to the group with the isolates from Rio Grande do Norte (group 3, BS 95%). The only exception was isolate 04, the most distant from the others, occurring at the base of the isolates group and PMeV-ES (BS 100%). The PMeV2-ES and the isolate 04 showed great genetic divergence compared to the others.
The genetic diversity study of PMeV from different Brazilian states, including Bahia, Espírito Santo, Pernambuco, Ceará, and Rio Grande do Norte, verified levels of identity higher than 88% among the isolates, demonstrating that the region is conserved, even among isolates from distant regions (Daltro et al. 2014). The authors hypothesized that the lack of tendency of isolates from the same region to group was probably due to the repeated introduction of several virus strains over the years, due to the constant movement of viruses through infected seeds or seedlings or through the putative vector.
However, up to now, no evidence was obtained to prove this hypothesis. Recent studies have shown that PMeV-Mx can be transmitted through seeds (Tapia-Tussell et al. 2015). In other hand, papaya sticky disease seed transmission studies conducted so far in Brazil were not able to prove that PMeV/PMeV-2 was seed transmitted (Abreu et al. 2012, Meissner Filho et al. 2022).
The phylogenetic analysis carried out using ML analysis showed that the Brazilian isolates, except for PMeV2-RN, form a single highly supported monophyletic group (BS 100%) (Fig. 3b). This information was already expected in view of the high conservation among the isolates, which was evidenced by the high levels of identity, even among isolates from distant regions. Thus, the absence of variation among isolates resulted in a low phylogenetic signal. In addition, it is possible to observe that the isolates from the same state do not have a single origin, indicating that different strains of the virus are present in both Bahia and Rio Grande do Norte. Moreover, it was possible to verify a great phylogenetic proximity between the isolate from Espírito Santo (PMeV2-ES) and isolate 04, from Bahia (BS 93%) (Fig. 3B).
CONCLUSION
Sequence analysis of PMeV2-RN and other viruses available in the Genbank database had already indicated the lack of a closer phylogenetic relationship between PMEV2-RN and PMeV2-ES, which possibly indicates different virus lineage. The genetic diversity study revealed that the RpRd region was conserved among the different PMeV2 isolates. Knowing the diversity of PMeV2 that is associated with papaya sticky disease in Brazil is crucial for a better understanding of the pathosystem, and it may support the establishment of more efficient diagnostic and control methods for the disease. Also, the study of diversity, especially of a new virus associated with papaya sticky disease, is essential for a better understanding of the plant-pathogen interaction, as viral genomes are the key to understanding how viruses interact with the host cells.
ACKNOWLEDGMENTS
Not applicable.
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1
Miller, M. A., Pfeiffer, W. and Schwartz, T. (2010). Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In: Proceedings of the Gateway Computing Environments Workshop (GCE), (p. 1-8). New Orleans: IEEE.
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How to cite:
Cruz Neto, A. J., Souza, S. O., Andrade, E. C., Daltro, C. B., Oliveira, A. M. G., Barbosa, C. J. and Schnadelbach, A. S. (2025). Molecular characterization and genetic diversity of papaya meleira virus 2 associated with papaya sticky disease in northeast Brazil. Bragantia, 84, e20240252. https://doi.org/10.1590/1678-4499.20240252
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FUNDING
Fundação de Amparo à Pesquisa do Estado da BahiaGrant No.: 238/2014Coordenação de Aperfeicoamento de Pessoal de Nível SuperiorFinance code 001
DATA AVAILABILITY STATEMENT
Data are available from the corresponding author upon reasonable request.
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Edited by
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Section Editor:
Robson Di Piero https://orcid.org/0000-0002-2897-2890
PMeV2: papaya meleira virus 2.