Genetic Differentiation and Migration Fluxes of Viruses from Melon Crops and Crop Edge Weeds

Abstract

Weeds surrounding crops may act as alternative hosts, playing important epidemiological roles as virus reservoirs and impacting virus evolution. We used high-throughput sequencing to identify viruses in Spanish melon crops and plants belonging to three pluriannual weed species, Ecballium elaterium, Malva sylvestris, and Solanum nigrum, sampled at the edges of the crops. Melon and E. elaterium, both belonging to the family Cucurbitaceae, shared three virus species, whereas there was no virus species overlap between melon and the other two weeds. The diversity of cucurbit aphid-borne yellows virus (CABYV) and tomato leaf curl New Delhi virus (ToLCNDV), both in melon and E. elaterium, was further studied by amplicon sequencing. Phylogenetic and population genetics analyses showed that the CABYV population was structured by the host, identifying three sites in the CABYV RNA-dependent RNA polymerase under positive selection, perhaps reflecting host adaptation. The ToLCNDV population was much less diverse than the CABYV one, likely as a consequence of the relatively recent introduction of ToLCNDV in Spain. In spite of its low diversity, we identified geographical but no host differentiation for ToLCNDV. Potential virus migration fluxes between E. elaterium and melon plants were also analyzed. For CABYV, no evidence of migration between the populations of the two hosts was found, whereas important fluxes were identified between geographically distant subpopulations for each host. For ToLCNDV, in contrast, evidence of migration from melon to E. elaterium was found, but not the other way around. IMPORTANCE It has been reported that about half of the emerging diseases affecting plants are caused by viruses. Alternative hosts often play critical roles in virus emergence as virus reservoirs, bridging host species that are otherwise unconnected and/or favoring virus diversification. In spite of this, the viromes of potential alternative hosts remain largely unexplored. In the case of crops, pluriannual weeds at the crop edges may play these roles. Here, we took advantage of the power of high-throughput sequencing to characterize the viromes of three weed species frequently found at the edges of melon crops. We identified three viruses shared by melon and the cucurbit weed, with two of them being epidemiologically relevant for melon crops. Further genetic analyses showed that these two viruses had contrasting patterns of diversification and migration, providing an interesting example on the role that weeds may play in the ecology and evolution of viruses affecting crops.

Keywords: CABYV; CCYV; CmEV; ToLCNDV; genetic differentiation; high-throughput sequencing; migration fluxes; virus reservoirs.

FIG 1

Map showing the sampling regions and the location of the melon fields. The sampling regions (Murcia and Castilla-La Mancha, Spain) are highlighted in gray. The sites, from where the samples of melon and associated weeds were collected, are represented using the different symbols shown in the legend.

FIG 2

Phylogenetic relationships between different isolates of cucurbit aphid-borne yellows virus (CABYV) inferred from partial sequences of open reading frame (ORF) 2. Minimum evolution trees were inferred using the MEGA X program using the K2P substitution model following a Gamma distribution. A first tree was inferred using 60 Spanish isolates characterized in this study (A); the cluster formed by CABYV isolates from E. elaterium is highlighted in gray. The sequence of a French isolate, CABYV-N (NC003688), was used as an outgroup. A second tree was inferred by incorporating a total of 86 CABYV isolates from Asia and Spain downloaded from the NCBI database (B). The arrows show host-specific intercrossing. First letters denote the host species: M, melon; EE, E. elaterium; W, watermelon; C, Cucumis sativus; Cu, Cucurbitaceae; MC, Momordica charantia; Sq, squash. The number in the format X.Y indicates the site of collection and sample number (e.g., M/3.150 is sample 150 collected from site 3). The scale bar corresponds to 0.01 nucleotide substitutions per site. Bootstrap values (1,000 pseudoreplicates) > 65% are shown.

FIG 3

Genetic diversity of cucurbit aphid-borne yellows virus (CABYV) isolates in mean posterior nonsynonymous (β) and synonymous (α) positions at each codon of the open reading frame (ORF) 2 for isolates from melon (A) and E. elaterium (B). Differences between the estimated diversity values (β-α) are represented in the horizontal axis. Lines marked with arrows represent sites under positive selection detected by the FUBAR method implemented in the Datamonkey web server with a posterior probability value of 0.9.

FIG 4

Diffusion pathway of Cucurbit aphid-borne yellows virus (CABYV) among melon crops and crop edge weeds in sites 1, 2, 3, and 4. Thickness of lines represents supported migration rates with a mean indicator of >0.5: solid black arrows, very strong support with BF >15; dashed black arrows, strong support 3 < BF < 15. The Murcia region (Spain) is shown in gray. White circles show the sites from where CABYV was isolated from both hosts, melon and E. elaterium. Yellow and green circles represent CABYV populations from melon and E. elaterium, respectively, in each of the sites.

FIG 5

Phylogenetic tree of Spanish tomato leaf curl New Delhi virus (ToLCNDV) isolates inferred from the full sequence of the AV2 gene. Minimum evolution trees were inferred from an alignment incorporating the 60 isolates from this study and another 77 Spanish isolates from the NCBI database with the MEGA X program using the K2P substitution model. First letters denote the host species: M, melon; EE, E. elaterium, C: Cucumis sativus; Cu, Cucurbitaceae; D, Datura stramonium and T, Tomato. The number in the format X.Y indicates the collection site and the sample number (e.g., M/3.150 is sample 150 collected from site 3). The scale bar corresponds to 0.001 nucleotide substitutions per site. Bootstrap values (1,000 pseudoreplicates) > 65% are shown.

FIG 6

Diffusion pathway of Tomato yellow leaf curl New Delhi virus (ToLCNDV) among melon crops and crop edge weeds in sites 2, 3, and 4. Thickness of lines represents supported migration rates with a mean indicator of >0.5: solid black arrows, very strong support with BF >15; dashed black arrows, strong support 3 < BF < 15. The Murcia region (Spain) is shown in gray. White circles show the sites from where ToLCNDV was isolated. Yellow and green circles represent ToLCNDV populations from melon and E. elaterium, respectively, in each of the sites.