Dynamics of the rice yellow mottle disease in western Burkina Faso: Epidemic monitoring, spatio-temporal variation of viral diversity, and pathogenicity in a disease hotspot.

The rice yellow mottle virus (RYMV) is a model in plant virus molecular epidemiology, with the reconstruction of historical introduction routes at the scale of the African continent. However, information on patterns of viral prevalence and viral diversity over multiple years at a local scale remains scarce, in spite of potential implications for crop protection. Here, we describe a 5-year (2015-9) monitoring of RYMV prevalence in six sites from western Burkina Faso (geographic areas of Bama, Banzon, and Karfiguela). It confirmed one irrigated site as a disease hotspot and also found one rainfed lowland (RL) site with occasional high prevalence levels. Within the studied fields, a pattern of disease aggregation was evidenced at a 5-m distance, as expected for a mechanically transmitted virus. Next, we monitored RYMV genetic diversity in the irrigated disease hotspot site, revealing a high viral diversity, with the current coexistence of various distinct genetic groups at the site scale (ca. 520 ha) and also within various specific fields (25 m side). One genetic lineage, named S1bzn, is the most recently emerged group and increased in frequency over the studied period (from 20 per cent or less in 2015-6 to more than 65 per cent in 2019). Its genome results from a recombination between two other lineages (S1wa and S1ca). Finally, experimental work revealed that three rice varieties commonly cultivated in Burkina Faso were not different in terms of resistance level, and we also found no significant effect of RYMV genetic groups on symptom expression and viral load. We found, however, that infection outcome depended on the specific RYMV isolate, with two isolates from the lineage S1bzn accumulating at the highest level at early infections. Overall, this study documents a case of high viral prevalence, high viral diversity, and co-occurrence of divergent genetic lineages at a small geographic scale. A recently emerged lineage, which comprises viral isolates inducing severe symptoms and high accumulation under controlled conditions, could be recently rising through natural selection. Following up the monitoring of RYMV diversity is required to confirm this trend and further understand the factors driving the local maintenance of viral diversity.

Role of Acrostyle Cuticular Proteins in the Retention of an Aphid Salivary Effector.

To avoid the activation of plant defenses and ensure sustained feeding, aphids are assumed to use their mouthparts to deliver effectors into plant cells. A recent study has shown that effectors detected near feeding sites are differentially distributed in plant tissues. However, the precise process of effector delivery into specific plant compartments is unknown. The acrostyle, a cuticular organ located at the tip of maxillary stylets that transiently binds plant viruses via its stylin proteins, may participate in this specific delivery process. Here, we demonstrate that Mp10, a saliva effector released into the plant cytoplasm during aphid probing, binds to the acrostyles of Acyrthosiphon pisum and Myzus persicae. The effector probably interacts with Stylin-03 as a lowered Mp10-binding to the acrostyle was observed upon RNAi-mediated reduction in Stylin-03 production. In addition, Stylin-03 and Stylin-01 RNAi aphids exhibited changes in their feeding behavior as evidenced by electrical penetration graph experiments showing longer aphid probing behaviors associated with watery saliva release into the cytoplasm of plant cells. Taken together, these data demonstrate that the acrostyle also has effector binding capacity and supports its role in the delivery of aphid effectors into plant cells.

Characterizing viral within-host diversity in fast and non-equilibrium demo-genetic dynamics.

High-throughput sequencing has opened the route for a deep assessment of within-host genetic diversity that can be used, e.g., to characterize microbial communities and to infer transmission links in infectious disease outbreaks. The performance of such characterizations and inferences cannot be analytically assessed in general and are often grounded on computer-intensive evaluations. Then, being able to simulate within-host genetic diversity across time under various demo-genetic assumptions is paramount to assess the performance of the approaches of interest. In this context, we built an original model that can be simulated to investigate the temporal evolution of genotypes and their frequencies under various demo-genetic assumptions. The model describes the growth and the mutation of genotypes at the nucleotide resolution conditional on an overall within-host viral kinetics, and can be tuned to generate fast non-equilibrium demo-genetic dynamics. We ran simulations of this model and computed classic diversity indices to characterize the temporal variation of within-host genetic diversity (from high-throughput amplicon sequences) of virus populations under three demographic kinetic models of viral infection. Our results highlight how demographic (viral load) and genetic (mutation, selection, or drift) factors drive variations in within-host diversity during the course of an infection. In particular, we observed a non-monotonic relationship between pathogen population size and genetic diversity, and a reduction of the impact of mutation on diversity when a non-specific host immune response is activated. The large variation in the diversity patterns generated in our simulations suggests that the underlying model provides a flexible basis to produce very diverse demo-genetic scenarios and test, for instance, methods for the inference of transmission links during outbreaks.

Synergy between an emerging monopartite begomovirus and a DNA-B component.

In recent decades, a legion of monopartite begomoviruses transmitted by the whitefly Bemisia tabaci has emerged as serious threats to vegetable crops in Africa. Recent studies in Burkina Faso (West Africa) reported the predominance of pepper yellow vein Mali virus (PepYVMLV) and its frequent association with a previously unknown DNA-B component. To understand the role of this DNA-B component in the emergence of PepYVMLV, we assessed biological traits related to virulence, virus accumulation, location in the tissue and transmission. We demonstrate that the DNA-B component is not required for systemic movement and symptom development of PepYVMLV (non-strict association), but that its association produces more severe symptoms including growth arrest and plant death. The increased virulence is associated with a higher viral DNA accumulation in plant tissues, an increase in the number of contaminated nuclei of the phloem parenchyma and in the transmission rate by B. tabaci. Our results suggest that the association of a DNA-B component with the otherwise monopartite PepYVMLV is a key factor of its emergence.

Correction: Estimation of the dispersal distances of an aphid-borne virus in a patchy landscape.

[This corrects the article DOI: 10.1371/journal.pcbi.1006085.].

Management of yellow dwarf disease in Europe in a post-neonicotinoid agriculture.

Barley/cereal yellow dwarf viruses (YDVs) cause yellow dwarf disease (YDD), which is a continuous risk to cereals production worldwide. These viruses cause leaf yellowing and stunting, resulting in yield reductions of up to 80%. YDVs have been a consistent but low-level problem in European cereal cultivation for the last three decades, mostly due to the availability of several effective insecticides (largely pyrethroids and more recently neonicotinoids) against aphid vectors. However, this has changed recently, with many insecticides being lost, culminating in a recent European Union (EU) regulation prohibiting outdoor use of the neonicotinoid-insecticide compounds. This change is coupled with the growing challenge of insecticide-resistant aphids, the lack of genetic resources against YDVs, and a knowledge deficit around the parameters responsible for the emergence and spread of YDD. This means that economic sustainability of cereal cultivation in several European countries including France and United Kingdom is now again threatened by this aphid-vectored viral disease. In this review, we summarize the current knowledge on the YDV pathosystem, describe management options against YDD, analyse the impacts of the neonicotinoid ban in Europe, and consider future strategies to control YDV. © 2020 Society of Chemical Industry.

Multi-scale spatial genetic structure of the vector-borne pathogen 'Candidatus Phytoplasma prunorum' in orchards and in wild habitats.

Inferring the dispersal processes of vector-borne plant pathogens is a great challenge because the plausible epidemiological scenarios often involve complex spread patterns at multiple scales. The spatial genetic structure of 'Candidatus Phytoplasma prunorum', responsible for European stone fruit yellows disease, was investigated by the application of a combination of statistical approaches to genotype data of the pathogen sampled from cultivated and wild compartments in three French Prunus-growing regions. This work revealed that the prevalence of the different genotypes is highly uneven both between regions and compartments. In addition, we identified a significant clustering of similar genotypes within a radius of 50 km or less, but not between nearby wild and cultivated Prunus. We also provide evidence that infected plants are transferred between production areas, and that both species of the Cacopsylla pruni complex can spread the pathogen. Altogether, this work supports a main epidemiological scenario where 'Ca. P. prunorum' is endemic in - and generally acquired from - wild Prunus by its immature psyllid vectors. The latter then migrate to shelter plants that epidemiologically connect sites less than 50 km apart by later providing infectious mature psyllids to their "migration basins". Such multi-scale studies could be useful for other pathosystems.

Analyzing the Influence of Landscape Aggregation on Disease Spread to Improve Management Strategies.

Epidemiological models are increasingly used to predict epidemics and improve management strategies. However, they rarely consider landscape characteristics although such characteristics can influence the epidemic dynamics and, thus, the effectiveness of disease management strategies. Here, we present a generic in silico approach which assesses the influence of landscape aggregation on the costs associated with an epidemic and on improved management strategies. We apply this approach to sharka, one of the most damaging diseases of Prunus trees, for which a management strategy is already applied in France. Epidemic simulations were carried out with a spatiotemporal stochastic model under various management strategies in landscapes differing in patch aggregation. Using sensitivity analyses, we highlight the impact of management parameters on the economic output of the model. We also show that the sensitivity analysis can be exploited to identify several strategies that are, according to the model, more profitable than the current French strategy. Some of these strategies are specific to a given aggregation level, which shows that management strategies should generally be tailored to each specific landscape. However, we also identified a strategy that is efficient for all levels of landscape aggregation. This one-size-fits-all strategy has important practical implications because of its simple applicability at a large scale.

Improving Management Strategies of Plant Diseases Using Sequential Sensitivity Analyses.

Improvement of management strategies of epidemics is often hampered by constraints on experiments at large spatiotemporal scales. A promising approach consists of modeling the biological epidemic process and human interventions, which both impact disease spread. However, few methods enable the simultaneous optimization of the numerous parameters of sophisticated control strategies. To do so, we propose a heuristic approach (i.e., a practical improvement method approximating an optimal solution) based on sequential sensitivity analyses. In addition, we use an economic improvement criterion based on the net present value, accounting for both the cost of the different control measures and the benefit generated by disease suppression. This work is motivated by sharka (caused by Plum pox virus), a vector-borne disease of prunus trees (especially apricot, peach, and plum), the management of which in orchards is mainly based on surveillance and tree removal. We identified the key parameters of a spatiotemporal model simulating sharka spread and control and approximated optimal values for these parameters. The results indicate that the current French management of sharka efficiently controls the disease, but it can be economically improved using alternative strategies that are identified and discussed. The general approach should help policy makers to design sustainable and cost-effective strategies for disease management.

Turnip Mosaic Virus Is a Second Example of a Virus Using Transmission Activation for Plant-to-Plant Propagation by Aphids.

Cauliflower mosaic virus (CaMV; family Caulimoviridae) responds to the presence of aphid vectors on infected plants by forming specific transmission morphs. This phenomenon, coined transmission activation (TA), controls plant-to-plant propagation of CaMV. A fundamental question is whether other viruses rely on TA. Here, we demonstrate that transmission of the unrelated turnip mosaic virus (TuMV; family Potyviridae) is activated by the reactive oxygen species H2O2 and inhibited by the calcium channel blocker LaCl3 H2O2-triggered TA manifested itself by the induction of intermolecular cysteine bonds between viral helper component protease (HC-Pro) molecules and by the formation of viral transmission complexes, composed of TuMV particles and HC-Pro that mediates vector binding. Consistently, LaCl3 inhibited intermolecular HC-Pro cysteine bonds and HC-Pro interaction with viral particles. These results show that TuMV is a second virus using TA for transmission but using an entirely different mechanism than CaMV. We propose that TuMV TA requires reactive oxygen species (ROS) and calcium signaling and that it is operated by a redox switch.IMPORTANCE Transmission activation, i.e., a viral response to the presence of vectors on infected hosts that regulates virus acquisition and thus transmission, is an only recently described phenomenon. It implies that viruses contribute actively to their transmission, something that has been shown before for many other pathogens but not for viruses. However, transmission activation has been described so far for only one virus, and it was unknown whether other viruses also rely on transmission activation. Here we present evidence that a second virus uses transmission activation, suggesting that it is a general transmission strategy.

Functional Transcomplementation between Wheat Dwarf Virus Strains in Wheat and Barley.

Wheat dwarf virus, transmitted by the leafhopper Psammotettix alienus in a persistent, non-propagative manner, infects numerous species from the Poaceae family. Data associated with wheat dwarf virus (WDV) suggest that some isolates preferentially infect wheat while other preferentially infect barley. This allowed to define the wheat strain and the barley strain. There are contradictory results in the literature regarding the ability of each of these two strains to infect its non-preferred host. To improve knowledge on the interactions between WDV strains and barley and wheat, transmission experiments were carried out using barcoded P. alienus and an experimental design based on single/sequential acquisitions of WDV strains and on transmissions to wheat and barley. Results showed that (I) WDV strains are transmitted with similar efficiencies by P. alienus males, females and larvae, (II) WDV wheat and barley strains do not infect barley and wheat plants, respectively, and (III) a functional transcomplementation between the wheat and barley strains allows a mixed infection of barley and wheat. The described ability of each WDV strain to infect a non-host plant in the presence of the other viral strain must be considered to analyze data available on WDV host range.

Small Bottleneck Size in a Highly Multipartite Virus during a Complete Infection Cycle.

Multipartite viruses package their genomic segments independently and thus incur the risk of being unable to transmit their entire genome during host-to-host transmission if they undergo severe bottlenecks. In this paper, we estimated the bottleneck size during one infection cycle of Faba bean necrotic stunt virus (FBNSV), an octopartite nanovirus whose segments have been previously shown to converge to particular and unequal relative frequencies within host plants and aphid vectors. Two methods were used to derive this estimate, one based on the probability of transmission of the virus and the other based on the temporal evolution of the relative frequency of markers for two genomic segments, one frequent and one rare (segment N and S, respectively), both in plants and vectors. Our results show that FBNSV undergoes severe bottlenecks during aphid transmission. Further, even though the bottlenecks are always narrow under our experimental conditions, they slightly widen with the number of transmitting aphids. In particular, when several aphids are used for transmission, the bottleneck size of the segments is also affected by within-plant processes and, importantly, significantly differs across segments. These results indicate that genetic drift not only must be an important process affecting the evolution of these viruses but also that these effects vary across genomic segments and, thus, across viral genes, a rather unique and intriguing situation. We further discuss the potential consequences of our findings for the transmission of multipartite viruses.IMPORTANCE Multipartite viruses package their genomic segments in independent capsids. The most obvious cost of such genomic structure is the risk of losing at least one segment during host-to-host transmission. A theoretical study has shown that for nanoviruses, composed of 6 to 8 segments, hundreds of copies of each segment need to be transmitted to ensure that at least one copy of each segment was present in the host. These estimations seem to be very high compared to the size of the bottlenecks measured with other viruses. Here, we estimated the bottleneck size during one infection cycle of FBNSV, an octopartite nanovirus. We show that these bottlenecks are always narrow (few viral particles) and slightly widen with the number of transmitting aphids. These results contrast with theoretical predictions and illustrate the fact that a new conceptual framework is probably needed to understand the transmission of highly multipartite viruses.

Estimation of the dispersal distances of an aphid-borne virus in a patchy landscape.

Characterising the spatio-temporal dynamics of pathogens in natura is key to ensuring their efficient prevention and control. However, it is notoriously difficult to estimate dispersal parameters at scales that are relevant to real epidemics. Epidemiological surveys can provide informative data, but parameter estimation can be hampered when the timing of the epidemiological events is uncertain, and in the presence of interactions between disease spread, surveillance, and control. Further complications arise from imperfect detection of disease and from the huge number of data on individual hosts arising from landscape-level surveys. Here, we present a Bayesian framework that overcomes these barriers by integrating over associated uncertainties in a model explicitly combining the processes of disease dispersal, surveillance and control. Using a novel computationally efficient approach to account for patch geometry, we demonstrate that disease dispersal distances can be estimated accurately in a patchy (i.e. fragmented) landscape when disease control is ongoing. Applying this model to data for an aphid-borne virus (Plum pox virus) surveyed for 15 years in 605 orchards, we obtain the first estimate of the distribution of flight distances of infectious aphids at the landscape scale. About 50% of aphid flights terminate beyond 90 m, which implies that most infectious aphids leaving a tree land outside the bounds of a 1-ha orchard. Moreover, long-distance flights are not rare-10% of flights exceed 1 km. By their impact on our quantitative understanding of winged aphid dispersal, these results can inform the design of management strategies for plant viruses, which are mainly aphid-borne.

Using sensitivity analysis to identify key factors for the propagation of a plant epidemic.

Identifying the key factors underlying the spread of a disease is an essential but challenging prerequisite to design management strategies. To tackle this issue, we propose an approach based on sensitivity analyses of a spatiotemporal stochastic model simulating the spread of a plant epidemic. This work is motivated by the spread of sharka, caused by plum pox virus, in a real landscape. We first carried out a broad-range sensitivity analysis, ignoring any prior information on six epidemiological parameters, to assess their intrinsic influence on model behaviour. A second analysis benefited from the available knowledge on sharka epidemiology and was thus restricted to more realistic values. The broad-range analysis revealed that the mean duration of the latent period is the most influential parameter of the model, whereas the sharka-specific analysis uncovered the strong impact of the connectivity of the first infected orchard. In addition to demonstrating the interest of sensitivity analyses for a stochastic model, this study highlights the impact of variation ranges of target parameters on the outcome of a sensitivity analysis. With regard to sharka management, our results suggest that sharka surveillance may benefit from paying closer attention to highly connected patches whose infection could trigger serious epidemics.

Exploiting Genetic Information to Trace Plant Virus Dispersal in Landscapes.

During the past decade, knowledge of pathogen life history has greatly benefited from the advent and development of molecular epidemiology. This branch of epidemiology uses information on pathogen variation at the molecular level to gain insights into a pathogen's niche and evolution and to characterize pathogen dispersal within and between host populations. Here, we review molecular epidemiology approaches that have been developed to trace plant virus dispersal in landscapes. In particular, we highlight how virus molecular epidemiology, nourished with powerful sequencing technologies, can provide novel insights at the crossroads between the blooming fields of landscape genetics, phylogeography, and evolutionary epidemiology. We present existing approaches and their limitations and contributions to the understanding of plant virus epidemiology.

Rapid accumulation and low degradation: key parameters of Tomato yellow leaf curl virus persistence in its insect vector Bemisia tabaci.

Of worldwide economic importance, Tomato yellow leaf curl virus (TYLCV, Begomovirus) is responsible for one of the most devastating plant diseases in warm and temperate regions. The DNA begomoviruses (Geminiviridae) are transmitted by the whitefly species complex Bemisia tabaci. Although geminiviruses have long been described as circulative non-propagative viruses, observations such as long persistence of TYLCV in B. tabaci raised the question of their possible replication in the vector. We monitored two major TYLCV strains, Mild (Mld) and Israel (IL), in the invasive B. tabaci Middle East-Asia Minor 1 cryptic species, during and after the viral acquisition, within two timeframes (0-144 hours or 0-20 days). TYLCV DNA was quantified using real-time PCR, and the complementary DNA strand of TYLCV involved in viral replication was specifically quantified using anchored real-time PCR. The DNA of both TYLCV strains accumulated exponentially during acquisition but remained stable after viral acquisition had stopped. Neither replication nor vertical transmission were observed. In conclusion, our quantification of the viral loads and complementary strands of both Mld and IL strains of TYLCV in B. tabaci point to an efficient accumulation and preservation mechanism, rather than to a dynamic equilibrium between replication and degradation.

Assessing the Mismatch Between Incubation and Latent Periods for Vector-Borne Diseases: The Case of Sharka.

The relative durations of the incubation period (the time between inoculation and symptom expression) and of the latent period (the time between inoculation and infectiousness of the host) are poorly documented for plant diseases. However, the extent of asynchrony between the ends of these two periods (i.e., their mismatch) can be a key determinant of the epidemic dynamics for many diseases and consequently it is of primary interest in the design of disease management strategies. In order to assess this mismatch, an experimental approach was developed and applied using sharka, a severe disease caused by Plum pox virus (PPV, genus Potyvirus, family Potyviridae) affecting trees belonging to the genus Prunus. Leaves of infected young peach trees were used individually as viral sources in aphid-mediated transmission tests carried out at different time points postinoculation in order to bracket symptom onset. By fitting a nonlinear logistic model to the obtained transmission rates, we demonstrated that the first symptoms appear on leaves 1 day before they rapidly become infectious. In addition, among symptomatic leaves, symptom intensity and transmission rate are positively correlated. These results strengthen the conclusion that, under our experimental conditions, incubation and latent periods of PPV infection are almost synchronous.

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.

Sharka epidemiology and worldwide management strategies: learning lessons to optimize disease control in perennial plants.

Many plant epidemics that cause major economic losses cannot be controlled with pesticides. Among them, sharka epidemics severely affect prunus trees worldwide. Its causal agent, Plum pox virus (PPV; genus Potyvirus), has been classified as a quarantine pathogen in numerous countries. As a result, various management strategies have been implemented in different regions of the world, depending on the epidemiological context and on the objective (i.e., eradication, suppression, containment, or resilience). These strategies have exploited virus-free planting material, varietal improvement, surveillance and removal of trees in orchards, and statistical models. Variations on these management options lead to contrasted outcomes, from successful eradication to widespread presence of PPV in orchards. Here, we present management strategies in the light of sharka epidemiology to gain insights from this worldwide experience. Although focused on sharka, this review highlights more general levers and promising approaches to optimize disease control in perennial plants.