Transcriptome and small RNAome profiling uncovers how a recombinant begomovirus evades RDRγ-mediated silencing of viral genes and outcompetes its parental virus in mixed infection.

Tomato yellow leaf curl virus (TYLCV, genus Begomovirus, family Geminiviridae) causes severe disease of cultivated tomatoes. Geminiviruses replicate circular single-stranded genomic DNA via rolling-circle and recombination-dependent mechanisms, frequently generating recombinants in mixed infections. Circular double-stranded intermediates of replication also serve as templates for Pol II bidirectional transcription. IS76, a recombinant derivative of TYLCV with a short sequence in the bidirectional promoter/origin-of-replication region acquired from a related begomovirus, outcompetes TYLCV in mixed infection and breaks disease resistance in tomato Ty-1 cultivars. Ty-1 encodes a γ-clade RNA-dependent RNA polymerase (RDRγ) implicated in Dicer-like (DCL)-mediated biogenesis of small interfering (si)RNAs directing gene silencing. Here, we profiled transcriptome and small RNAome of Ty-1 resistant and control susceptible plants infected with TYLCV, IS76 or their combination at early and late infection stages. We found that RDRγ boosts production rates of 21, 22 and 24 nt siRNAs from entire genomes of both viruses and modulates DCL activities in favour of 22 and 24 nt siRNAs. Compared to parental TYLCV, IS76 undergoes faster transition to the infection stage favouring rightward transcription of silencing suppressor and coat protein genes, thereby evading RDRγ activity and facilitating its DNA accumulation in both single and mixed infections. In coinfected Ty-1 plants, IS76 efficiently competes for host replication and transcription machineries, thereby impairing TYLCV replication and transcription and forcing its elimination associated with further increased siRNA production. RDRγ is constitutively overexpressed in Ty-1 plants, which correlates with begomovirus resistance, while siRNA-generating DCLs (DCL2b/d, DCL3, DCL4) and genes implicated in siRNA amplification (α-clade RDR1) and function (Argonaute2) are upregulated to similar levels in TYLCV- and IS76-infected susceptible plants. Collectively, IS76 recombination facilitates replication and promotes expression of silencing suppressor and coat proteins, which allows the recombinant virus to evade the negative impact of RDRγ-boosted production of viral siRNAs directing transcriptional and posttranscriptional silencing.

Refining the emergence scenario of the invasive recombinant Tomato yellow leaf curl virus -IS76.

TYLCV-IS76, a unique recombinant between tomato yellow leaf curl virus (TYLCV) and tomato yellow leaf curl Sardinia virus (TYLCSV), has replaced its parental viruses in southern Morocco. To refine its emergence scenario, its fitness was monitored experimentally in conditions aiming at reproducing natural situations, i.e. superinfection of plants already infected with parental viruses and competition with other TYLCV/TYLCSV recombinants (LSRec) automatically generated in plants coinfected with TYLCV and TYLCSV. TYLCV-IS76 accumulated significantly more than parental viruses regardless of plant age and superinfection delay. Although TYLCV-IS76 and LSRec both accumulated more than parental viruses in laboratory conditions, LSRec were displaced by TYLCV-IS76 in nature like parental viruses were. TYLCV-IS76 did not exhibit any vector transmission advantage over LSRec and TYLCV the most competitive parental virus. Thus, it is apparently only in the plant compartment that the recombination event that generated TYLCV-IS76, induced the competitiveness advantage by which the last became first.

Alfalfa leaf curl virus is efficiently acquired by its aphid vector Aphis craccivora but inefficiently transmitted.

Alfalfa leaf curl virus (ALCV) is the first geminivirus for which aphid transmission was reported. Transmission by Aphis craccivora was determined previously to be highly specific and circulative. Using various complementary techniques, the transmission journey of ALCV was monitored from its uptake from infected plant tissues up to the head of its vector. ALCV was shown to be restricted to phloem tissues using fluorescence in situ hybridization (FISH) and electropenetrography (EPG) monitoring of virus acquisition. Furthermore, the virus is heterogeneously distributed in phloem tissues, as revealed by FISH and quantitative PCR of viral DNA acquired by EPG-monitored aphids. Despite the efficient ingestion of viral DNA, about 106 viral DNA copies per insect in a 15 h feeding period on ALCV-infected plants, the individual maximum transmission rate was 12 %. Transmission success was related to a critical viral accumulation, around 1.6×107 viral DNA copies per insect, a threshold that generally needed more than 48 h to be reached. Moreover, whereas the amount of acquired virus did not decrease over time in the whole aphid body, it declined in the haemolymph and heads. ALCV was not detected in progenies of viruliferous aphids and did not affect aphid fitness. Compared to geminiviruses transmitted by whiteflies or leafhoppers, or to luteoviruses transmitted by aphids, the transmission efficiency of ALCV by A. craccivora is low. This result is discussed in relation to the aphid vector of this geminivirus and the agroecological features of alfalfa, a hardy perennial host plant.

Co-Acquired Nanovirus and Geminivirus Exhibit a Contrasted Localization within Their Common Aphid Vector.

Single-stranded DNA (ssDNA) plant viruses belong to the families Geminiviridae and Nanoviridae. They are transmitted by Hemipteran insects in a circulative, mostly non-propagative, manner. While geminiviruses are transmitted by leafhoppers, treehoppers, whiteflies and aphids, nanoviruses are transmitted exclusively by aphids. Circulative transmission involves complex virus-vector interactions in which epithelial cells have to be crossed and defense mechanisms counteracted. Vector taxa are considered a relevant taxonomic criterion for virus classification, indicating that viruses can evolve specific interactions with their vectors. Thus, we predicted that, although nanoviruses and geminiviruses represent related viral families, they have evolved distinct interactions with their vector. This prediction is also supported by the non-structural Nuclear Shuttle Protein (NSP) that is involved in vector transmission in nanoviruses but has no similar function in geminiviruses. Thanks to the recent discovery of aphid-transmitted geminiviruses, this prediction could be tested for the geminivirus alfalfa leaf curl virus (ALCV) and the nanovirus faba bean necrotic stunt virus (FBNSV) in their common vector, Aphis craccivora. Estimations of viral load in midgut and head of aphids, precise localization of viral DNA in cells of insect vectors and host plants, and virus transmission tests revealed that the pathway of the two viruses across the body of their common vector differs both quantitatively and qualitatively.

Fitness advantage of inter-species TYLCV recombinants induced by beneficial intra-genomic interactions rather than by specific mutations.

Tomato yellow leaf curl virus (TYLCV) and its related viruses are prone to recombination. It was reported that random homologous recombination between 20% diverging TYLCV related species is rarely deleterious and may be associated with a fitness advantage. Indeed, TYLCV-IS76, a recombinant between the 20% divergent TYLCV and tomato yellow leaf curl Sardinia virus (TYLCSV), exhibited a higher fitness than that of parental viruses. As this typical fitness advantage was observed with TYLCV-IS76 representatives of different pedigrees, it was thought that it is induced by beneficial intra-genomic interactions rather than by specific mutations. This hypothesis was further supported with TYLCV-IS141, a TYLCV recombinant with a short TYLCSV inherited fragment of around 141 nts, slightly longer than that of TYLCV-IS76. Indeed, the typical fitness advantage was detected irrespective of the position of the recombination breakpoint (loci 76 or 141) and the sequences of the TYLCV and TYLCSV inherited fragments.

A multicellular way of life for a multipartite virus.

A founding paradigm in virology is that the spatial unit of the viral replication cycle is an individual cell. Multipartite viruses have a segmented genome where each segment is encapsidated separately. In this situation the viral genome is not recapitulated in a single virus particle but in the viral population. How multipartite viruses manage to efficiently infect individual cells with all segments, thus with the whole genome information, is a long-standing but perhaps deceptive mystery. By localizing and quantifying the genome segments of a nanovirus in host plant tissues we show that they rarely co-occur within individual cells. We further demonstrate that distinct segments accumulate independently in different cells and that the viral system is functional through complementation across cells. Our observation deviates from the classical conceptual framework in virology and opens an alternative possibility (at least for nanoviruses) where the infection can operate at a level above the individual cell level, defining a viral multicellular way of life.

Characterization of Begomoviruses Sampled during Severe Epidemics in Tomato Cultivars Carrying the Ty-1 Gene.

Tomato yellow leaf curl virus (TYLCV, genus Begomovirus, family Geminiviridae) is a major species that causes a tomato disease for which resistant tomato hybrids (mainly carriers of the Ty-1/Ty-3 gene) are being used widely. We have characterized begomoviruses severely affecting resistant tomato crops in Southeast Spain. Circular DNA was prepared from samples by rolling circle amplification, and sequenced by massive sequencing (2015) or cloning and Sanger sequencing (2016). Thus, 23 complete sequences were determined, all belonging to the TYLCV Israel strain (TYLCV-IL). Massive sequencing also revealed the absence of other geminiviral and beta-satellite sequences. A phylogenetic analysis showed that the Spanish isolates belonged to two groups, one related to early TYLCV-IL isolates in the area (Group 1), and another (Group 2) closely related to El Jadida (Morocco) isolates, suggesting a recent introduction. The most parsimonious evolutionary scenario suggested that the TYLCV isolates of Group 2 are back recombinant isolates derived from TYLCV-IS76, a recombinant virus currently predominating in Moroccan epidemics. Thus, an infectious Group 2 clone (TYLCV-Mu15) was constructed and used in in planta competition assays against TYLCV-IS76. TYLCV-Mu15 predominated in single infections, whereas TYLCV-IS76 did so in mixed infections, providing credibility to a scenario of co-occurrence of both types of isolates.

World Management of Geminiviruses.

Management of geminiviruses is a worldwide challenge because of the widespread distribution of economically important diseases caused by these viruses. Regardless of the type of agriculture, management is most effective with an integrated pest management (IPM) approach that involves measures before, during, and after the growing season. This includes starting with resistant cultivars and virus- and vector-free transplants and propagative plants. For high value vegetables, protected culture (e.g., greenhouses and screenhouses) allows for effective management but is limited owing to high cost. Protection of young plants in open fields is provided by row covers, but other measures are typically required. Measures that are used for crops in open fields include roguing infected plants and insect vector management. Application of insecticide to manage vectors (whiteflies and leafhoppers) is the most widely used measure but can cause undesirable environmental and human health issues. For annual crops, these measures can be more effective when combined with host-free periods of two to three months. Finally, given the great diversity of the viruses, their insect vectors, and the crops affected, IPM approaches need to be based on the biology and ecology of the virus and vector and the crop production system. Here, we present the general measures that can be used in an IPM program for geminivirus diseases, specific case studies, and future challenges.

Accumulation and transmission of alphasatellite, betasatellite and tomato yellow leaf curl virus in susceptible and Ty-1-resistant tomato plants.

Begomoviruses (family Geminiviridae) are frequently associated with alphasatellites and betasatellites in the Old World. Tomato yellow leaf curl virus, one of the most damaging begomovirus species worldwide, was recently found associated with betasatellites in the eastern coast of the Mediterranean Sea, and in the Middle East region. Tomato yellow leaf curl virus (TYLCV)/betasatellite associations were shown to increase TYLCV virulence in experimental conditions. The sustainability of TYLCV/satellite associations in tomato was assessed here by estimating accumulation levels of satellites in comparison to TYLCV, vector transmission efficiency, and by testing how far the popular Ty-1 resistance gene used in most TYLCV-resistant tomato cultivars in the Mediterranean Basin is effective against betasatellites. Three satellites previously isolated from okra in Burkina Faso-of the species Cotton leaf curl Gezira betasatellite, Cotton leaf curl Gezira alphasatellite and Okra leaf curl Burkina Faso alphasatellite-were shown to accumulate at levels similar to, or higher than, the helper virus TYLCV-Mld in tomato plants from 32 to 150 days post inoculation (dpi). Cotton leaf curl Gezira betasatellite (CLCuGB) reduced TYLCV-Mld accumulation whereas alphasatellites did not. Transmission tests were performed with B. tabaci from plants infected with TYLCV-Mld/CLCuGB- or TYLCV-Mld/Okra leaf curl Burkina Faso alphasatellite. At 32 dpi, both satellites were transmitted to more than 50% of TYLCV-infected test plants. Betasatellite transmission, tested further with 150 dpi source plants was successful in more than 30% of TYLCV-infected test plants. Ty-1 resistant tomato plants co-infected with TYLCV (-Mld or -IL) and CLCuGB exhibited mild leaf curling and mosaic symptoms at the early stage of infection associated with a positive effect on TYLCV-IL accumulation, while resistant plants infected with TYLCV only, were asymptomatic. Together with previous experimental studies, these results further emphasize the potential risk of betasatellites to tomato cultivation, including with Ty-1 resistant cultivars.

Geometagenomics illuminates the impact of agriculture on the distribution and prevalence of plant viruses at the ecosystem scale.

Disease emergence events regularly result from human activities such as agriculture, which frequently brings large populations of genetically uniform hosts into contact with potential pathogens. Although viruses cause nearly 50% of emerging plant diseases, there is little systematic information about virus distribution across agro-ecological interfaces and large gaps in understanding of virus diversity in nature. Here we applied a novel landscape-scale geometagenomics approach to examine relationships between agricultural land use and distributions of plant-associated viruses in two Mediterranean-climate biodiversity hotspots (Western Cape region of South Africa and Rhône river delta region of France). In total, we analysed 1725 geo-referenced plant samples collected over two years from 4.5 × 4.5 km2 grids spanning farmlands and adjacent uncultivated vegetation. We found substantial virus prevalence (25.8-35.7%) in all ecosystems, but prevalence and identified family-level virus diversity were greatest in cultivated areas, with some virus families displaying strong agricultural associations. Our survey revealed 94 previously unknown virus species, primarily from uncultivated plants. This is the first effort to systematically evaluate plant-associated viromes across broad agro-ecological interfaces. Our findings indicate that agriculture substantially influences plant virus distributions and highlight the extent of current ignorance about the diversity and roles of viruses in nature.

Complete genome sequences of cowpea polerovirus 1 and cowpea polerovirus 2 infecting cowpea plants in Burkina Faso.

The full-length genome sequences of two novel poleroviruses found infecting cowpea plants, cowpea polerovirus 1 (CPPV1) and cowpea polerovirus 2 (CPPV2), were determined using overlapping RT-PCR and RACE-PCR. Whereas the 5845-nt CPPV1 genome was most similar to chickpea chlorotic stunt virus (73% identity), the 5945-nt CPPV2 genome was most similar to phasey bean mild yellow virus (86% identity). The CPPV1 and CPPV2 genomes both have a typical polerovirus genome organization. Phylogenetic analysis of the inferred P1-P2 and P3 amino acid sequences confirmed that CPPV1 and CPPV2 are indeed poleroviruses. Four apparently unique recombination events were detected within a dataset of 12 full polerovirus genome sequences, including two events in the CPPV2 genome. Based on the current species demarcation criteria for the family Luteoviridae, we tentatively propose that CPPV1 and CPPV2 should be considered members of novel polerovirus species.

The non-canonical tomato yellow leaf curl virus recombinant that displaced its parental viruses in southern Morocco exhibits a high selective advantage in experimental conditions.

Recombination events are frequently inferred from the increasing number of sequenced viral genomes, but their impact on natural viral populations has rarely been evidenced. TYLCV-IS76 is a recombinant (Begomovirus,Geminiviridae) between the Israel strain of tomato yellow leaf curl virus (TYLCV-IL) and the Spanish strain of tomato yellow leaf curl Sardinia virus (TYLCSV-ES) that was generated most probably in the late 1990s in southern Morocco (Souss). Its emergence in the 2000s coincided with the increasing use of resistant tomato cultivars bearing the Ty-1 gene, and led eventually to the entire displacement of both parental viruses in the Souss. Here, we provide compelling evidence that this viral population shift was associated with selection of TYLCV-IS76 viruses in tomato plants and particularly in Ty-1-bearing cultivars. Real-time quantitative PCR (qPCR) monitoring revealed that TYLCV-IS76 DNA accumulation in Ty-1-bearing plants was significantly higher than that of representatives of the parental virus species in single infection or competition assays. This advantage of the recombinant in Ty-1-bearing plants was not associated with a fitness cost in a susceptible, nearly isogenic, cultivar. In competition assays in the resistant cultivar, the DNA accumulation of the TYLCV-IL clone - the parent less affected by the Ty-1 gene in single infection - dropped below the qPCR detection level at 120 days post-infection (p.i.) and below the whitefly vector (Bemisia tabaci) transmissibility level at 60 days p.i. The molecular basis of the selective advantage of TYLCV-IS76 is discussed in relation to its non-canonical recombination pattern, and the RNA-dependent RNA polymerase encoded by the Ty-1 gene.

Metagenomic-Based Screening and Molecular Characterization of Cowpea-Infecting Viruses in Burkina Faso.

Cowpea, (Vigna unguiculata L. (Walp)) is an annual tropical grain legume. Often referred to as "poor man's meat", cowpea is one of the most important subsistence legumes cultivated in West Africa due to the high protein content of its seeds. However, African cowpea production can be seriously constrained by viral diseases that reduce yields. While twelve cowpea-infecting viruses have been reported from Africa, only three of these have so-far been reported from Burkina Faso. Here we use a virion-associated nucleic acids (VANA)-based metagenomics method to screen for the presence of cowpea viruses from plants collected from the three agro-climatic zones of Burkina Faso. Besides the three cowpea-infecting virus species which have previously been reported from Burkina Faso (Cowpea aphid borne mosaic virus [Family Potyviridae], the Blackeye cowpea mosaic virus-a strain of Bean common mosaic virus-[Family Potyviridae] and Cowpea mottle virus [Family Tombusviridae]) five additional viruses were identified: Southern cowpea mosaic virus (Sobemovirus genus), two previously uncharacterised polerovirus-like species (Family Luteoviridae), a previously uncharacterised tombusvirus-like species (Family Tombusviridae) and a previously uncharacterised mycotymovirus-like species (Family Tymoviridae). Overall, potyviruses were the most prevalent cowpea viruses (detected in 65.5% of samples) and the Southern Sudan zone of Burkina Faso was found to harbour the greatest degrees of viral diversity and viral prevalence. Partial genome sequences of the two novel polerovirus-like and tombusvirus-like species were determined and RT-PCR primers were designed for use in Burkina Faso to routinely detect all of these cowpea-associated viruses.

Mapping genetic determinants of viral traits with FST and quantitative trait locus (QTL) approaches.

The genetic determinism of viral traits can generally be dissected using either forward or reverse genetics because the clonal reproduction of viruses does not require the use of approaches based on laboratory crosses. Nevertheless, we hypothesized that recombinant viruses could be analyzed as sexually reproducing organisms, using either a quantitative trait loci (QTL) approach or a locus-by-locus fixation index (FST). Locus-by-locus FST analysis, and four different regressions and interval mapping algorithms of QTL analysis were applied to a phenotypic and genotypic dataset previously obtained from 47 artificial recombinant genomes generated between two begomovirus species. Both approaches assigned the determinant of within-host accumulation-previously identified using standard virology approaches-to a region including the 5׳ end of the replication-associated protein (Rep) gene and the upstream intergenic region. This study provides a proof of principle that QTL and population genetics tools can be extended to characterize the genetic determinants of viral traits.

Alfalfa Leaf Curl Virus: an Aphid-Transmitted Geminivirus.

The family Geminiviridae comprises seven genera differentiated by genome organization, sequence similarity, and insect vector. Capulavirus, an eighth genus, has been proposed to accommodate two newly discovered highly divergent geminiviruses that presently have no known vector. Alfalfa leaf curl virus, identified here as a third capulavirus, is shown to be transmitted by Aphis craccivora. This is the first report of an aphid-transmitted geminivirus.

The genomes of many yam species contain transcriptionally active endogenous geminiviral sequences that may be functionally expressed.

Endogenous viral sequences are essentially 'fossil records' that can sometimes reveal the genomic features of long extinct virus species. Although numerous known instances exist of single-stranded DNA (ssDNA) genomes becoming stably integrated within the genomes of bacteria and animals, there remain very few examples of such integration events in plants. The best studied of these events are those which yielded the geminivirus-related DNA elements found within the nuclear genomes of various Nicotiana species. Although other ssDNA virus-like sequences are included within the draft genomes of various plant species, it is not entirely certain that these are not contaminants. The Nicotiana geminivirus-related DNA elements therefore remain the only definitively proven instances of endogenous plant ssDNA virus sequences. Here, we characterize two new classes of endogenous plant virus sequence that are also apparently derived from ancient geminiviruses in the genus Begomovirus. These two endogenous geminivirus-like elements (EGV1 and EGV2) are present in the Dioscorea spp. of the Enantiophyllum clade. We used fluorescence in situ hybridization to confirm that the EGV1 sequences are integrated in the D. alata genome and showed that one or two ancestral EGV sequences likely became integrated more than 1.4 million years ago during or before the diversification of the Asian and African Enantiophyllum Dioscorea spp. Unexpectedly, we found evidence of natural selection actively favouring the maintenance of EGV-expressed replication-associated protein (Rep) amino acid sequences, which clearly indicates that functional EGV Rep proteins were probably expressed for prolonged periods following endogenization. Further, the detection in D. alata of EGV gene transcripts, small 21-24 nt RNAs that are apparently derived from these transcripts, and expressed Rep proteins, provides evidence that some EGV genes are possibly still functionally expressed in at least some of the Enantiophyllum clade species.

Genetic structure of Bemisia tabaci Med populations from home-range countries, inferred by nuclear and cytoplasmic markers: impact on the distribution of the insecticide resistance genes.

BACKGROUND: Insecticide resistance management in Bemisia tabaci is one of the main issues facing agricultural production today. An extensive survey was undertaken in five Mediterranean countries to examine the resistance status of Med B. tabaci species in its range of geographic origin and the relationship between population genetic structure and the distribution of resistance genes. The investigation combined molecular diagnostic tests, sequence and microsatellite polymorphism studies and monitoring of endosymbionts. RESULTS: High frequencies of pyrethroid (L925I and T929V, VGSC gene) and organophosphate (F331W, ace1 gene) resistance mutations were found in France, Spain and Greece, but not in Morocco or Tunisia. Sequence analyses of the COI gene delineated two closely related mitochondrial groups (Q1 and Q2), which were found either sympatrically (Spain) or separately (France). Only Q1 was observed in Greece, Morocco and Tunisia. Bayesian analyses based on microsatellite loci revealed three geographically delineated genetic groups (France, Spain, Morocco/Greece/Tunisia) and high levels of genetic differentiation even between neighbouring samples. Evidence was also found for hybridisation and asymmetrical gene flow between Q1 and Q2. CONCLUSIONS: Med B. tabaci is more diverse and structured than reported so far. On a large geographic scale, resistance is affected by population genetic structure, whereas on a local scale, agricultural practices appear to play a major role.

Identification and characterisation of a highly divergent geminivirus: evolutionary and taxonomic implications.

During a large scale "non a priori" survey in 2010 of South African plant-infecting single stranded DNA viruses, a highly divergent geminivirus genome was isolated from a wild spurge, Euphorbia caput-medusae. In addition to being infectious in E. caput-medusae, the cloned viral genome was also infectious in tomato and Nicotiana benthamiana. The virus, named Euphorbia caput-medusae latent virus (EcmLV) due to the absence of infection symptoms displayed by its natural host, caused severe symptoms in both tomato and N. benthamiana. The genome organisation of EcmLV is unique amongst geminiviruses and it likely expresses at least two proteins without any detectable homologues within public sequence databases. Although clearly a geminivirus, EcmLV is so divergent that we propose its placement within a new genus that we have tentatively named Capulavirus. Using a set of highly divergent geminiviruses genomes, it is apparent that recombination has likely been a primary process in the genus-level diversification of geminiviruses. It is also demonstrated how this insight, taken together with phylogenetic analyses of predicted coat protein and replication associated protein (Rep) amino acid sequences indicate that the most recent common ancestor of the geminiviruses was likely a dicot-infecting virus that, like modern day mastreviruses and becurtoviruses, expressed its Rep from a spliced complementary strand transcript.

Within-host dynamics of the emergence of Tomato yellow leaf curl virus recombinants.

Tomato yellow leaf curl virus (TYLCV) is a highly damaging begomovirus native to the Middle East. TYLCV has recently spread worldwide, recombining with other begomoviruses. Recent analysis of mixed infections between TYLCV and Tomato leaf curl Comoros begomovirus (ToLCKMV) has shown that, although natural selection preserves certain co-evolved intra-genomic interactions, numerous and diverse recombinants are produced at 120 days post-inoculation (dpi), and recombinant populations from different tomato plants are very divergent. Here, we investigate the population dynamics that lead to such patterns in tomato plants co-infected with TYLCV and ToLCKMV either by agro-inoculation or using the natural whitefly vector Bemisia tabaci. We monitored the frequency of parental and recombinant genotypes independently in 35 plants between 18 and 330 dpi and identified 177 recombinants isolated at different times. Recombinants were detected from 18 dpi and their frequency increased over time to reach about 50% at 150 dpi regardless of the inoculation method. The distribution of breakpoints detected on 96 fully sequenced recombinants was consistent with a continuous generation of new recombinants as well as random and deterministic effects in their maintenance. A severe population bottleneck of around 10 genomes was estimated during early systemic infection-a phenomenon that could account partially for the heterogeneity in recombinant patterns observed among plants. The detection of the same recombinant genome in six of the thirteen plants analysed beyond 30 dpi supported the influence of selection on observed recombination patterns. Moreover, a highly virulent recombinant genotype dominating virus populations within one plant has, apparently, the potential to be maintained in the natural population according to its infectivity, within-host accumulation, and transmission efficiency - all of which were similar or intermediate to those of the parent genotypes. Our results anticipate the outcomes of natural encounters between TYLCV and ToLCKMV.