Genetic Diversity, Evolution, and Diagnosis of Sugarcane Yellow Leaf Virus from 19 Sugarcane-Producing Locations Worldwide.

Sugarcane yellow leaf virus (SCYLV), the causal agent of yellow leaf, has been reported in an increasing number of sugarcane-growing locations since its first report in the 1990s in Brazil, Florida, and Hawaii. In this study, the genetic diversity of SCYLV was investigated using the genome coding sequence (5,561 to 5,612 nt) of 109 virus isolates from 19 geographical locations, including 65 new isolates from 16 geographical regions worldwide. These isolates were distributed in three major phylogenetic lineages (BRA, CUB, and REU), except for one isolate from Guatemala. Twenty-two recombination events were identified among the 109 isolates of SCYLV, thus confirming that recombination was a significant driving force in the genetic diversity and evolution of this virus. No temporal signal was found in the genomic sequence dataset, most likely because of the short temporal window of the 109 SCYLV isolates (1998 to 2020). Among 27 primers reported in the literature for the detection of the virus by RT-PCR, none matched 100% with all 109 SCYLV sequences, suggesting that the use of some primer pairs may not result in the detection of all virus isolates. Primers YLS111/YLS462, which were the first primer pair used by numerous research organizations to detect the virus by RT-PCR, failed to detect isolates belonging to the CUB lineage. In contrast, primer pair ScYLVf1/ScYLVr1 efficiently detected isolates of all three lineages. Continuous pursuit of knowledge of SCYLV genetic variability is therefore critical for effective diagnosis of yellow leaf, especially in virus-infected and mainly asymptomatic sugarcane plants.

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.].

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.

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.

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.

Fighting Sharka in Peach: Current Limitations and Future Perspectives.

Sharka, caused by Plum Pox Virus (PPV), is by far the most important infectious disease of peach [P. persica (L.) Batsch] and other Prunus species. The progressive spread of the virus in many important growing areas throughout Europe poses serious issues to the economic sustainability of stone fruit crops, peach in particular. The adoption of internationally agreed-upon rules for diagnostic tests, strain-specific monitoring schemes and spatial-temporal modeling of virus spread, are all essential for a more effective sharka containment. The EU regulations on nursery activity should be modified based on the zone delimitation of PPV presence, limiting open-field production of propagation materials only to virus-free areas. Increasing the efficiency of preventive measures should be augmented by the short-term development of resistant cultivars. Putative sources of resistance/tolerance have been recently identified in peach germplasm, although the majority of novel resistant sources to PPV-M have been found in almond. However, the complexity of introgression from related-species imposes the search for alternative strategies. The use of genetic engineering, particularly RNA interference (RNAi)-based approaches, appears as one of the most promising perspectives to introduce a durable resistance to PPV in peach germplasm, notwithstanding the well-known difficulties of in vitro plant regeneration in this species. In this regard, rootstock transformation to induce RNAi-mediated systemic resistance would avoid the transformation of numerous commercial cultivars, and may alleviate consumer resistance to the use of GM plants.

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.

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.

SNaPshot and CE-SSCP: Two Simple and Cost-Effective Methods to Reveal Genetic Variability Within a Virus Species.

The multiplex SNaPshot and the capillary electrophoresis-single-strand conformation polymorphism (CE-SSCP) procedures are here used for rapid and high-throughput description of the molecular variability of viral populations. Both approaches are based on (1) standard amplification of genomic sequence(s), (2) labeled primers or labeled single-stranded DNA, and (3) migration of fluorescent-labeled molecules in capillary electrophoresis system. The SNaPshot technology was used to describe the diversity of 20 targeted single nucleotide polymorphisms (SNPs) selected from alignment of viral genomic sequences retrieved from public database. The CE-SSCP procedure was applied to identify the polymorphisms of two small (<500 bases in length) genomic regions of viral genomes. The different steps of SNaPshot and CE-SSCP setup procedures are presented using Potato virus Y (PVY, Potyvirus) and Plum pox virus (PPV, Potyvirus) RNA viruses as molecular targets, respectively.

Metagenomics Approaches Based on Virion-Associated Nucleic Acids (VANA): An Innovative Tool for Assessing Without A Priori Viral Diversity of Plants.

This chapter describes an efficient approach that combines quality and yield extraction of viral nucleic acids from plants containing high levels of secondary metabolites and a sequence-independent amplification procedure for both the inventory of known plant viruses and the discovery of unknown ones. This approach turns out to be a useful tool for assessing the virome (the genome of all the viruses that inhabit a particular organism) of plants of interest. We here show that this approach enables the identification of a novel Potyvirus member within a single plant already known to be infected by two other Potyvirus species.

Mediterranean and central-eastern European countries host viruses of two different clades of plum pox virus strain M.

The genetic diversity of plum pox virus strain M (PPV-M) was assessed by analyzing 28 isolates collected in 8 European countries. Two genomic fragments spanning the (Cter)P3-6K1-(Nter)CI coding region as well as the full coat protein coding region were sequenced directly from PCR products. Phylogenetic analysis showed that the geographical origin of the collected isolates was clearly associated with two different PPV-M clades. Moreover, the pattern of substitutions in the CP gene shed light on the evolutionary relationships between PPV-M and the recombinant strains PPV-Rec and PPV-T.

Investigating Disease Spread between Two Assessment Dates with Permutation Tests on a Lattice.

ABSTRACT Mapping and analyzing the disease status of individual plants within a study area at successive dates can give insight into the processes involved in the spread of a disease. We propose a permutation method to analyze such spatiotemporal maps of binary data (healthy or diseased plants) in regularly spaced plantings. It requires little prior information on the causes of disease spread and handles missing plants and censored data. A Monte Carlo test is used to assess whether the location of newly diseased plants is independent of the location of previously diseased plants. The test takes account of the significant spatial structures at each date in order to separate nonrandomness caused by the structure at one date from nonrandomness caused by the dependence between newly diseased plants and previously diseased plants. If there is a nonrandom structure at both dates, independent patterns are simulated by randomly shifting the entire pattern observed at the second date. Otherwise, independent patterns are simulated by randomly reallocating the positions of one group of diseased plants. Simulated and observed patterns of disease are then compared through distance-based statistics. The performance of the method and its robustness are evaluated by its ability to accurately identify simulated independent and dependent bivariate point patterns. Additionally, two realworld spatiotemporal maps with contrasting disease progress illustrate how the tests can provide valuable clues about the processes of disease spread. This method can supplement biological investigations and be used as an exploratory step before developing a specific mechanistic model.

Factors Affecting the Spread of Plum pox virus Strain M in Peach Orchards Subjected to Roguing in France.

ABSTRACT We evaluated the impact of roguing on the spread and persistence of the aggressive Plum pox virus strain M (PPV-M) in 19 peach orchard blocks in Southern France. During a 7- to 10-year period, orchards were visually inspected for PPV symptoms, and symptomatic trees were removed every year. Disease incidence was low in all orchards at disease discovery and was <1% in 16 of the 19 orchard blocks. The spread of Sharka disease was limited in all 19 blocks, with an annual disease incidence between 2 and 6%. However, new symptomatic trees were continuously detected, even after 7 to 10 years of uninterrupted control measures. An extended Cox model was developed to evaluate to what extent tree location, orchard characteristics, environment, and disease status within the vicinity influenced the risk of infection through time. Eleven variables with potential effect on tree survival (i.e., maintenance of a tree in a disease- free status through time) were selected from survey data and databases created using a geographical information system. Area of the orchard, density of planting, distance of a tree from the edge of the orchard block sharing a boundary with another diseased orchard, and distance to the nearest previously detected symptomatic tree had a significant effect on the risk for a tree to become infected through time. The combined results of this study suggest that new PPV-M infections within orchards subjected to roguing resulted from exogenous sources of inoculum, disease development of latent infected trees, as well as infected trees overlooked within the orchards during visual surveys. A revision of the survey and the roguing procedures used for more effective removal of potential sources of inoculum within the orchards and in the vicinity of the orchards would improve disease control suppression of PPV.

Origin, world-wide dispersion, bio-geographical diversification, radiation and recombination: an evolutionary history of Yam mild mosaic virus (YMMV).

We developed an evolutionary epidemiological approach to understand the regional and world-wide dispersion of Yam mild mosaic virus (YMMV) by retracing its evolutionary history. Analyses of the distribution and the prevalence of YMMV in the Caribbean islands of Guadeloupe and Martinique, and in French Guyana revealed that YMMV has a wide repartition and different prevalence on Dioscorea alata L. (Asian and Oceanic origin), on D. cayenensis Lam.-D. rotundata Poir. (African origin) and on D. trifida L. (Amazon and the Caribbean origin) in this region. Considering the data on the current dispersion of the virus and the evolution and the history of the yams, the phylogenetic analysis of the 3' terminal part of the YMMV genome gave a consistent support of the Asian-Pacific origin of YMMV from D. alata species. The YMMV phylogenetic tree is star-like, suggesting an early split of the genetic lineages. An important part of the clades is constituted by a single lineage arisen by recombination. The largest emerging monophyletic group illustrates well YMMV geographical dispersion. This evolutionary pattern contrasts with the one revealed by the African distinct lineages and by the second significant monophyletic group, for which a host adaptation to D. trifida is suggested. The analysis of the pattern of nucleotide substitutions in the CP gene revealed that purifying selection dominates the evolution of the CP of potyviruses and strongly operates on the YMMV. Switching events, radiation, host and geographical adaptation and recombination events are proposed as major traits of YMMV evolutionary history.

Spatial Pattern Analysis of Sharka Disease (Plum pox virus Strain M) in Peach Orchards of Southern France.

ABSTRACT The spatial pattern of Sharka disease, caused by Plum pox virus (PPV) strain M, was investigated in 18 peach plots located in two areas of southern France. PPV infections were monitored visually for each individual tree during one to three consecutive years. Point pattern and correlation-type approaches were undertaken using the binary data directly or after parsing them in contiguous quadrats of 4, 9, and 16 trees. Ordinary runs generally revealed a low but variable proportion of rows with adjacent symptomatic trees. Aggregation of disease incidence was indicated by the theta parameter of the beta-binomial distribution and related indices in 15 of the 18 plots tested for at least one assessment date of each. When aggregation was detected, it was indicated at all quadrat sizes and tended to be a function of disease incidence, as shown by the binary form of Taylor's power law. Spatial analysis by distance indices (SADIE) showed a nonrandom arrangement of quadrats with infected trees in 14 plots. The detection of patch clusters enclosing quadrats with above-average density of symptomatic trees, ellipsoidal in shape and generally extending from 4 to 14 trees within rows and from 4 to 10 trees perpendicular to the rows, could be interpreted as local areas of influence of PPV spread. Spatial patterns at the plot scale were often characterized by the occurrence of several clusters of infected trees located up to 90 m apart in the direction of the rows. When several time assessments were available, increasing clustering over time was generally evidenced by stronger values of the clustering index and by increasing patch cluster size. The combination of the different approaches revealed a wide range of spatial patterns of PPV-M, from no aggregation to high aggregation of symptomatic trees at all spatial scales investigated. Such patterns suggested that aphid transmission to neighboring trees occurred frequently but was not systematic. The mechanism of primary virus introduction, the age and structure of the orchards when infected, and the diversity of vector species probably had a strong influence on the secondary spread of the disease. This study provides a more complete understanding of PPV-M patterns which could help to improve targeting of removal of PPV-infected trees for more effective disease control.