An Agent-Based Model Shows How Mixed Infections Drive Multiyear Pathotype Dynamics in a Plant-Virus System.

Mathematical models are widely used to understand the evolution and epidemiology of plant pathogens under a variety of scenarios. Here, we used this approach to analyze the effects of different traits intrinsic and extrinsic to plant-virus interactions on the dynamics of virus pathotypes in genetically heterogeneous plant-virus systems. For this, we propose an agent-based epidemiological model that includes epidemiologically significant pathogen life-history traits related to virulence, transmission, and survival in the environment and allows for integrating long- and short-distance transmission, primary and secondary infections, and within-host pathogen competition in mixed infections. The study focuses on the tobamovirus-pepper pathosystem. Model simulations allowed us to integrate pleiotropic effects of resistance-breaking mutations on different virus life-history traits into the net costs of resistance breaking, allowing for predictions on multiyear pathotype dynamics. We also explored the effects of two control measures, the use of host resistance and roguing of symptomatic plants, that modify epidemiological attributes of the pathogens to understand how their populations will respond to evolutionary pressures. One major conclusion points to the importance of pathogen competition within mixed-infected hosts as a component of the overall fitness of each pathogen that, thus, drives their multiyear dynamics.

Repeated loss of the ability of a wild pepper disease resistance gene to function at high temperatures suggests that thermoresistance is a costly trait.

Specificity in plant-pathogen gene-for-gene (GFG) interactions is determined by the recognition of pathogen proteins by the products of plant resistance (R) genes. The evolutionary dynamics of R genes in plant-virus systems is poorly understood. We analyse the evolution of the L resistance locus to tobamoviruses in the wild pepper Capsicum annuum var. glabriusculum (chiltepin), a crop relative undergoing incipient domestication. The frequency, and the genetic and phenotypic diversity, of the L locus was analysed in 41 chiltepin populations under different levels of human management over its distribution range in Mexico. The frequency of resistance was lower in Cultivated than in Wild populations. L-locus genetic diversity showed a strong spatial structure with no isolation-by-distance pattern, suggesting environment-specific selection, possibly associated with infection by the highly virulent tobamoviruses found in the surveyed regions. L alleles differed in recognition specificity and in the expression of resistance at different temperatures, broad-spectrum recognition of P0 + P1 pathotypes and expression above 32°C being ancestral traits that were repeatedly lost along L-locus evolution. Overall, loss of resistance co-occurs with incipient domestication and broad-spectrum resistance expressed at high temperatures has apparent fitness costs. These findings contribute to understand the role of fitness trade-offs in plant-virus coevolution.

Ecological Strategies for Resource Use by Three Bromoviruses in Anthropic and Wild Plant Communities.

Ecological strategies for resource utilisation are important features of pathogens, yet have been overshadowed by stronger interest in genetic mechanisms underlying disease emergence. The purpose of this study is to ask whether host range and transmission traits translate into ecological strategies for host-species utilisation in a heterogeneous ecosystem, and whether host utilisation corresponds to genetic differentiation among three bromoviruses. We combine high-throughput sequencing and population genomics with analyses of species co-occurrence to unravel the ecological strategies of the viruses across four habitat types. The results show that the bromoviruses that were more closely related genetically did not share similar ecological strategies, but that the more distantly related pair did. Shared strategies included a broad host range and more frequent co-occurrences, which both were habitat-dependent. Each habitat thus presents as a barrier to gene flow, and each virus has an ecological strategy to navigate limitations to colonising non-natal habitats. Variation in ecological strategies could therefore hold the key to unlocking events that lead to emergence.

Tobamoviruses Show Broad Host Ranges and Little Genetic Diversity Among Four Habitat Types of a Heterogeneous Ecosystem.

Host ranges of plant viruses are poorly known, as studies have focused on pathogenic viruses in crops and adjacent wild plants. High-throughput sequencing (HTS) avoids the bias toward plant-virus interactions that result in disease. Here we study the host ranges of tobamoviruses, important pathogens of crops, using HTS analyses of an extensive sample of plant communities in four habitats of a heterogeneous ecosystem. Sequences of 17 virus operational taxonomic units (OTUs) matched references in the Tobamovirus genus, eight had narrow host ranges, and five had wide host ranges. Regardless of host range, the OTU hosts belonged to taxonomically distant families, suggesting no phylogenetic constraints in host use associated with virus adaptation, and that tobamoviruses may be host generalists. The OTUs identified as tobacco mild green mosaic virus (TMGMV), tobacco mosaic virus (TMV), pepper mild mottle virus, and Youcai mosaic virus had the largest realized host ranges that occurred across habitats and exhibited host use unrelated to the degree of human intervention. This result is at odds with assumptions that contact-transmitted viruses would be more abundant in crops than in wild plant communities and could be explained by effective seed-, contact-, or pollinator-mediated transmission or by survival in the soil. TMGMV and TMV had low genetic diversity that was not structured according to habitat or host plant taxonomy, which indicated that phenotypic plasticity allows virus genotypes to infect new hosts with no need for adaptive evolution. Our results underscore the relevance of ecological factors in host range evolution, in addition to the more often studied genetic factors. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Reversion of a resistance-breaking mutation shows reversion costs and high virus diversity at necrotic local lesions.

An instance of host range evolution relevant to plant virus disease control is resistance breaking. Resistance breaking can be hindered by across-host fitness trade-offs generated by negative effects of resistance-breaking mutations on the virus fitness in susceptible hosts. Different mutations in pepper mild mottle virus (PMMoV) coat protein result in the breaking in pepper plants of the resistance determined by the L3 resistance allele. Of these, mutation M138N is widespread in PMMoV populations, despite associated fitness penalties in within-host multiplication and survival. The stability of mutation M138N was analysed by serial passaging in L3 resistant plants. Appearance on passaging of necrotic local lesions (NLL), indicating an effective L3 resistance, showed reversion to nonresistance-breaking phenotypes was common. Most revertant genotypes had the mutation N138K, which affects the properties of the virus particle, introducing a penalty of reversion. Hence, the costs of reversion may determine the evolution of resistance-breaking in addition to resistance-breaking costs. The genetic diversity of the virus population in NLL was much higher than in systemically infected tissues, and included mutations reported to break L3 resistance other than M138N. Infectivity assays on pepper genotypes with different L alleles showed high phenotypic diversity in respect to L alleles in NLL, including phenotypes not reported in nature. Thus, high diversity at NLL may potentiate the appearance of genotypes that enable the colonization of new host genotypes or species. Collectively, the results of this study contribute to better understanding the evolutionary dynamics of resistance breaking and host-range expansions.

Metagenomics show high spatiotemporal virus diversity and ecological compartmentalisation: Virus infections of melon, Cucumis melo, crops, and adjacent wild communities.

The emergence of viral diseases results from novel transmission dynamics between wild and crop plant communities. The bias of studies towards pathogenic viruses of crops has distracted from knowledge of non-antagonistic symbioses in wild plants. Here, we implemented a high-throughput approach to compare the viromes of melon (Cucumis melo) and wild plants of crop (Crop) and adjacent boundaries (Edge). Each of the 41-plant species examined was infected by at least one virus. The interactions of 104 virus operational taxonomic units (OTUs) with these hosts occurred largely within ecological compartments of either Crop or Edge, with Edge having traits of a reservoir community. Local scale patterns of infection were characterised by the positive correlation between plant and virus richness at each site, the tendency for increased specialist host use through seasons, and specialist host use by OTUs observed only in Crop, characterised local-scale patterns of infection. In this study of systematically sampled viromes of a crop and adjacent wild communities, most hosts showed no disease symptoms, suggesting non-antagonistic symbioses are common. The coexistence of viruses within species-rich ecological compartments of agro-systems might promote the evolution of a diversity of virus strategies for survival and transmission. These communities, including those suspected as reservoirs, are subject to sporadic changes in assemblages, and so too are the conditions that favour the emergence of disease.

Structuring of plant communities across agricultural landscape mosaics: the importance of connectivity and the scale of effect.

BACKGROUND: Plant communities of fragmented agricultural landscapes, are subject to patch isolation and scale-dependent effects. Variation in configuration, composition, and distance from one another affect biological processes of disturbance, productivity, and the movement ecology of species. However, connectivity and spatial structuring among these diverse communities are rarely considered together in the investigation of biological processes. Spatially optimised predictor variables that are based on informed measures of connectivity among communities, offer a solution to untangling multiple processes that drive biodiversity. RESULTS: To address the gap between theory and practice, a novel spatial optimisation method that incorporates hypotheses of community connectivity, was used to estimate the scale of effect of biotic and abiotic factors that distinguish plant communities. We tested: (1) whether different hypotheses of connectivity among sites was important to measuring diversity and environmental variation among plant communities; and (2) whether spatially optimised variables of species relative abundance and the abiotic environment among communities were consistent with diversity parameters in distinguishing four habitat types; namely Crop, Edge, Oak, and Wasteland. The global estimates of spatial autocorrelation, which did not consider environmental variation among sites, indicated significant positive autocorrelation under four hypotheses of landscape connectivity. The spatially optimised approach indicated significant positive and negative autocorrelation of species relative abundance at fine and broad scales, which depended on the measure of connectivity and environmental variation among sites. CONCLUSIONS: These findings showed that variation in community diversity parameters does not necessarily correspond to underlying spatial structuring of species relative abundance. The technique used to generate spatially-optimised predictors is extendible to incorporate multiple variables of interest along with a priori hypotheses of landscape connectivity. Spatially-optimised variables with appropriate definitions of connectivity might be better than diversity parameters in explaining functional differences among communities.

Population Genomics of Plant Viruses: The Ecology and Evolution of Virus Emergence.

The genomics era has revolutionized studies of adaptive evolution by monitoring large numbers of loci throughout the genomes of many individuals. Ideally, the investigation of emergence in plant viruses requires examining the population dynamics of both virus and host, their interactions with each other, with other organisms and the abiotic environment. Genetic mechanisms that affect demographic processes are now being studied with high-throughput technologies, traditional genetics methods, and new computational tools for big-data. In this review, we discuss the utility of these approaches to monitor and detect changes in virus populations within cells and individuals, and over wider areas across species and communities of ecosystems. The advent of genomics in virology has fostered a multidisciplinary approach to tackling disease risk. The ability to make sense of the information now generated in this integrated setting is by far the most substantial obstacle to the ultimate goal of plant virology to minimize the threats to food security posed by disease. To achieve this goal, it is imperative to understand and forecast how populations respond to future changes in complex natural systems.

Ecological fitting is the forerunner to diversification in a plant virus with broad host range.

The evolution and diversification of ssRNA plant viruses are often examined under reductionist conditions that ignore potentially much wider biotic interactions. The host range of a plant virus is central to interactions at higher levels that are organized by both fitness and ecological criteria. Here we employ a strategy to minimize sampling biases across distinct plant communities and combine it with a high-throughput sequencing approach to examine the influence of four habitats on the evolution of Watermelon mosaic virus (WMV). Local, regional and global levels of genetic diversity that correspond to spatial and temporal extents are used to infer haplotype relationships using network and phylogenetic approaches. We find that the incidence and genetic diversity of WMV were structured significantly by host species and habitat type. A single haplotype that infected 11 host species of a total of 24 showed that few constraints on host species use exist in the crop communities. When the evolution of WMV was examined at broader levels of organization, we found variation in genetic diversity and contrasting host use footprints that broadly corresponded to habitat effects. The findings demonstrated that nondeterministic ecological factors structured the genetic diversity of WMV. Habitat-driven constraints underlie host use preferences.

Coexistence of nestedness and modularity in host-pathogen infection networks.

The long-term coevolution of hosts and pathogens in their environment forms a complex web of multi-scale interactions. Understanding how environmental heterogeneity affects the structure of host-pathogen networks is a prerequisite for predicting disease dynamics and emergence. Although nestedness is common in ecological networks, and theory suggests that nested ecosystems are less prone to dynamic instability, why nestedness varies in time and space is not fully understood. Many studies have been limited by a focus on single habitats and the absence of a link between spatial variation and structural heterogeneity such as nestedness and modularity. Here we propose a neutral model for the evolution of host-pathogen networks in multiple habitats. In contrast to previous studies, our study proposes that local modularity can coexist with global nestedness, and shows that real ecosystems are found in a continuum between nested-modular and nested networks driven by intraspecific competition. Nestedness depends on neutral mechanisms of community assembly, whereas modularity is contingent on local adaptation and competition. The structural pattern may change spatially and temporally but remains stable over evolutionary timescales. We validate our theoretical predictions with a longitudinal study of plant-virus interactions in a heterogeneous agricultural landscape.

Evolution of plant-virus interactions: host range and virus emergence.

Changes in host range are central to virus emergence. Host range, together with its evolution, is determined by virus intrinsic factors, such as genetic traits determining its fitness in different hosts. Experimental analyses have shown the relevance in host range evolution of across-host fitness trade-offs. Host range is also determined by ecological factors extrinsic to the virus such as the distribution, abundance, and interaction of species, and understanding their role in host range evolution is a current challenge. Indeed, intrinsic and extrinsic factors, and the complexity of biotic and abiotic interactions, must be considered in order to provide generalisations on patterns of transmission, host range evolution, and disease emergence. This exciting new field of research is still in its infancy.

Coinfection Organizes Epidemiological Networks of Viruses and Hosts and Reveals Hubs of Transmission.

Multiple virus infections affect the competence of host plants to transmit disease. The effects of coinfection on transmission are expected to produce ecologically complex pathogen and host-pathogen interactions. However, the prediction of disease risk will rely on untangling nonrandom from random patterns of infection to identify underlying processes that drive these interactions. Are the spatial distributions of infections in complex multispecies systems random or not? For the first time, we use an empirical evaluation of this basic but nontrivial question to test the hypothesis that coinfection contributes to (i) nonrandom ecological interactions between hosts and viruses and (ii) structuring infection distributions. We use a novel approach that decomposed the ecological interactions of 11 generalist viruses in 47 host species in four habitats of an agroecosystem into single-infection and coinfection "modes." Then, we relate ecological structuring in infection networks to the distribution of infection using generalized regression models. The network analyses of coinfection showed that virus-host interactions occurred more often than expected at random in one of the four habitats, Edge. A pattern of specific interactions was shared between Edge and the ecosystem, indicating scale invariance. The regression modeling also showed that the plant community characteristics of Edge were unique in explaining infection distributions. The results showed that the spatial distribution of infection at the ecosystem level was not only a species-specific phenomenon but also, strongly structured by specific virus-virus and host-virus interactions. The evidence of scale invariance and the special role of Edge as a reservoir suggest that ecological interactions were less strongly structured by community differences among habitats than by wider-scale processes and traits underlying the interactions. Addressing whether reservoir communities significantly contribute to epidemiological processes at the ecosystem scale is a promising avenue for future research.

Tobamoviruses as Models for the Study of Virus Evolution.

The study of tobacco mosaic virus and other tobamovirus species has greatly contributed to the development of all areas of virology, including virus evolution. Research with tobamoviruses has been pioneer, or particularly significant, in all major areas of research in this field, including: the characterization of the genetic diversity of virus populations, the mechanisms and rates of generation of genetic diversity, the analysis of the genetic structure of virus populations and of the factors that shape it, the adaptation of viruses to hosts and the evolution of host range, and the evolution of virus taxa and of virus-host interactions. Many of these continue to be hot topics in evolutionary biology, or have been identified recently as such, including (i) host-range evolution, (ii) predicting the overcoming of resistance in crops, (iii) trade-offs between virus life-history traits in virus evolution, and (iv) the codivergence of viruses and hosts at different taxonomical and spatial scales. Tobamoviruses may be particularly appropriate to address these topics with plant viruses, as they provide convenient experimental systems, and as the detailed knowledge on their molecular and structural biology allows the analysis of the mechanisms behind evolutionary processes. Also, the extensive information on parameters related to infection dynamics and population structure may facilitate the development of realistic models to predict virus evolution. Certainly, tobamoviruses will continue to be favorite system for the study of virus evolution.

Analysis of Fitness Trade-Offs in the Host Range Expansion of an RNA Virus, Tobacco Mild Green Mosaic Virus.

The acquisition of new hosts provides a virus with more opportunities for transmission and survival but may be limited by across-host fitness trade-offs. Major causes of across-host trade-offs are antagonistic pleiotropy, that is, host differential phenotypic effects of mutations, a Genotype x Environment interaction, and epistasis, a Genotype x Genotype interaction. Here, we analyze if there are trade-offs, and what are the causes, associated with the acquisition by tobacco mild green mosaic virus (TMGMV) of a new host. For this, the multiplication of sympatric field isolates of TMGMV from its wild reservoir host Nicotiana glauca and from pepper crops was quantified in the original and the heterologous hosts. TMGMV isolates from N. glauca were adapted to their host, but pepper isolates were not adapted to pepper, and the acquisition of this new host was associated with a fitness penalty in the original host. Analyses of the collection of field isolates and of mutant genotypes derived from biologically active cDNA clones showed a role of mutations in the coat protein and the 3' untranslated region in determining within-host virus fitness. Fitness depended on host-specific effects of these mutations, on the genetic background in which they occurred, and on higher-order interactions of the type Genotype x Genotype x Environment. These types of effects had been reported to generate across-host fitness trade-offs under experimental evolution. Our results show they may also operate in heterogeneous natural environments and could explain why pepper isolates were not adapted to pepper and their lower fitness in N. glaucaIMPORTANCE The acquisition of new hosts conditions virus epidemiology and emergence; hence it is important to understand the mechanisms behind host range expansion. Experimental evolution studies have identified antagonistic pleiotropy and epistasis as genetic mechanisms that limit host range expansion, but studies from virus field populations are few. Here, we compare the performance of isolates of tobacco mild green mosaic virus from its reservoir host, Nicotiana glauca, and its new host, pepper, showing that acquisition of a new host was not followed by adaptation to it but was associated with a fitness loss in the original host. Analysis of mutations determining host-specific virus multiplication identified antagonistic pleiotropy, epistasis, and host-specific epistasis as mechanisms generating across-host fitness trade-offs that may prevent adaptation to pepper and cause a loss of fitness in N. glauca Thus, mechanisms determining trade-offs, identified under experimental evolution, could also operate in the heterogeneous environment in which natural plant virus populations occur.

Ecological Complexity in Plant Virus Host Range Evolution.

The host range of a plant virus is the number of species in which it can reproduce. Most studies of plant virus host range evolution have focused on the genetics of host-pathogen interactions. However, the distribution and abundance of plant viruses and their hosts do not always overlap, and these spatial and temporal discontinuities in plant virus-host interactions can result in various ecological processes that shape host range evolution. Recent work shows that the distributions of pathogenic and resistant genotypes, vectors, and other resources supporting transmission vary widely in the environment, producing both expected and unanticipated patterns. The distributions of all of these factors are influenced further by competitive effects, natural enemies, anthropogenic disturbance, the abiotic environment, and herbivory to mention some. We suggest the need for further development of approaches that (i) explicitly consider resource use and the abiotic and biotic factors that affect the strategies by which viruses exploit resources; and (ii) are sensitive across scales. Host range and habitat specificity will largely determine which phyla are most likely to be new hosts, but predicting which host and when it is likely to be infected is enormously challenging because it is unclear how environmental heterogeneity affects the interactions of viruses and hosts.

Scale dependencies and generalism in host use shape virus prevalence.

Processes that generate the distribution of pathogens and their interactions with hosts are not insensitive to changes in spatial scale. Spatial scales and species traits are often selected intentionally, based on practical considerations, ignoring biases that the scale and type of observation may introduce. Specifically, these biases might change the interpretation of disease-diversity relationships that are reported as either 'dilution' or 'amplification' effects. Here, we combine field data of a host-pathogen community with empirical models to test the effects that (i) spatial scale and (ii) host range have on the relationship between plant-virus infection prevalence and diversity. We show that prevalence-diversity relationships are scale-dependent and can produce opposite effects associated with different habitats at sub-ecosystem scales. The total number of host species of each virus reflected generalism at the ecosystem scale. However, plasticity in host range resembled habitat-specific specialization and also changed model predictions. We show that habitat heterogeneity, ignored at larger (ecosystem) spatial scales, influences pathogen distributions. Hence, understanding disease distributions and the evolution of pathogens requires reconciling specific hypotheses of the study with an appropriate spatial scale, or scales, and consideration of traits, such as host range, that might strongly contribute to biotic interactions.

Pleiotropic Effects of Resistance-Breaking Mutations on Particle Stability Provide Insight into Life History Evolution of a Plant RNA Virus.

In gene-for-gene host-virus interactions, virus evolution to infect and multiply in previously resistant host genotypes, i.e., resistance breaking, is a case of host range expansion, which is predicted to be associated with fitness penalties. Negative effects of resistance-breaking mutations on within-host virus multiplication have been documented for several plant viruses. However, understanding virus evolution requires analyses of potential trade-offs between different fitness components. Here we analyzed whether coat protein (CP) mutations in Pepper mild mottle virus that break L-gene resistance in pepper affect particle stability and, thus, survival in the environment. For this purpose, CP mutations determining the overcoming of L 3 and L 4 resistance alleles were introduced in biologically active cDNA clones. The kinetics of the in vitro disassembly of parental and mutant particles were compared under different conditions. Resistance-breaking mutations variously affected particle stability. Structural analyses identified the number and type of axial and side interactions of adjacent CP subunits in virions, which explained differences in particle stability and contribute to understanding of tobamovirus disassembly. Resistance-breaking mutations also affected virus multiplication and virulence in the susceptible host, as well as infectivity. The sense and magnitude of the effects of resistance-breaking mutations on particle stability, multiplication, virulence, or infectivity depended on the specific mutation rather than on the ability to overcome the different resistance alleles, and effects on different traits were not correlated. Thus, the results do not provide evidence of links or trade-offs between particle stability, i.e., survival, and other components of virus fitness or virulence.IMPORTANCE The effect of survival on virus evolution remains underexplored, despite the fact that life history trade-offs may constrain virus evolution. We approached this topic by analyzing whether breaking of L-gene resistance in pepper by Pepper mild mottle virus, determined by coat protein (CP) mutations, is associated with reduced particle stability and survival. Resistance-breaking mutations affected particle stability by altering the interactions between CP subunits. However, the sense and magnitude of these effects were unrelated to the capacity to overcome different resistance alleles. Thus, resistance breaking was not traded with survival. Resistance-breaking mutations also affected virus fitness within the infected host, virulence, and infectivity in a mutation-specific manner. Comparison of the effects of CP mutations on these various traits indicates that there are neither trade-offs nor positive links between survival and other life history traits. These results demonstrate that trade-offs between life history traits may not be a general constraint in virus evolution.

Environmental heterogeneity and the evolution of plant-virus interactions: Viruses in wild pepper populations.

Understanding host-pathogen interactions requires analyses to address the multiplicity of scales in heterogeneous landscapes. Anthropogenic influence on plant communities, especially cultivation, is a major cause of environmental heterogeneity. We have approached the analysis of how environmental heterogeneity determines plant-virus interactions by studying virus infection in a wild plant currently undergoing incipient domestication, the wild pepper or chiltepin, across its geographical range in Mexico. We have shown previously that anthropogenic disturbance is associated with higher infection and disease risk, and with disrupted patterns of host and virus genetic spatial structure. We now show that anthropogenic factors, species richness, host genetic diversity and density in communities supporting chiltepin differentially affect infection risk according to the virus analysed. We also show that in addition to these factors, a broad range of abiotic and biotic variables meaningful to continental scales, have an important role on the risk of infection depending on the virus. Last, we show that natural virus infection of chiltepin plants in wild communities results in decreased survival and fecundity, hence negatively affecting fitness. This important finding paves the way for future studies on plant-virus co-evolution.

Mutations That Determine Resistance Breaking in a Plant RNA Virus Have Pleiotropic Effects on Its Fitness That Depend on the Host Environment and on the Type, Single or Mixed, of Infection.

UNLABELLED: Overcoming host resistance in gene-for-gene host-virus interactions is an important instance of host range expansion, which can be hindered by across-host fitness trade-offs. Trade-offs are generated by negative effects of host range mutations on the virus fitness in the original host, i.e., by antagonistic pleiotropy. It has been reported that different mutations in Pepper mild mottle virus (PMMoV) coat protein result in overcoming L-gene resistance in pepper. To analyze if resistance-breaking mutations in PMMoV result in antagonistic pleiotropy, all reported mutations determining the overcoming of L(3) and L(4) alleles were introduced in biologically active cDNA clones. Then, the parental and mutant virus genotypes were assayed in susceptible pepper genotypes with an L(+), L(1), or L(2) allele, in single and in mixed infections. Resistance-breaking mutations had pleiotropic effects on the virus fitness that, according to the specific mutation, the host genotype, and the type of infection, single or mixed with other virus genotypes, were antagonistic or positive. Thus, resistance-breaking mutations can generate fitness trade-offs both across hosts and across types of infection, and the frequency of host range mutants will depend on the genetic structure of the host population and on the frequency of mixed infections by different virus genotypes. Also, resistance-breaking mutations variously affected virulence, which may further influence the evolution of host range expansion. IMPORTANCE: A major cause of virus emergence is host range expansion, which may be hindered by across-host fitness trade-offs caused by negative pleiotropy of host range mutations. An important instance of host range expansion is overcoming host resistance in gene-for-gene plant-virus interactions. We analyze here if mutations in the coat protein of Pepper mild mottle virus determining L-gene resistance-breaking in pepper have associated fitness penalties in susceptible host genotypes. Results show that pleiotropic effects of resistance-breaking mutations on virus fitness depend on the specific mutation, the susceptible host genotype, and the type of infection, single or mixed, with other virus genotypes. Accordingly, resistance-breaking mutations can have negative, positive, or no pleiotropic effects on virus fitness. These results underscore the complexity of host range expansion evolution and, specifically, the difficulty of predicting the overcoming of resistance factors in crops.

Aphid vector population density determines the emergence of necrogenic satellite RNAs in populations of cucumber mosaic virus.

The satellite RNAs of cucumber mosaic virus (CMV) that induce systemic necrosis in tomato plants (N-satRNA) multiply to high levels in the infected host while severely depressing CMV accumulation and, hence, its aphid transmission efficiency. As N-satRNAs are transmitted into CMV particles, the conditions for N-satRNA emergence are not obvious. Model analyses with realistic parameter values have predicted that N-satRNAs would invade CMV populations only when transmission rates are high. Here, we tested this hypothesis experimentally by passaging CMV or CMV+N-satRNAs at low or high aphid densities (2 or 8 aphids/plant). As predicted, high aphid densities were required for N-satRNA emergence. The results showed that at low aphid densities, random effects due to population bottlenecks during transmission dominate the epidemiological dynamics of CMV/CMV+N-satRNA. The results suggest that maintaining aphid populations at low density will prevent the emergence of highly virulent CMV+N-satRNA isolates.