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.].

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.

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.

Molecular test to assign individuals within the Cacopsylla pruni complex.

Crop protection requires the accurate identification of disease vectors, a task that can be made difficult when these vectors encompass cryptic species. Here we developed a rapid molecular diagnostic test to identify individuals of Cacopsyllapruni (Scopoli, 1763) (Hemiptera: Psyllidae), the main vector of the European stone fruit yellows phytoplasma. This psyllid encompasses two highly divergent genetic groups that are morphologically similar and that are characterized by genotyping several microsatellite markers, a costly and time-consuming protocol. With the aim of developing species-specific PCR primers, we sequenced the Internal Transcribed Spacer 2 (ITS2) on a collection of C. pruni samples from France and other European countries. ITS2 sequences showed that the two genetic groups represent two highly divergent clades. This enabled us to develop specific primers for the assignment of individuals to either genetic group in a single PCR, based on ITS2 amplicon size. All previously assigned individuals yielded bands of expected sizes, and the PCR proved efficient on a larger sample of 799 individuals. Because none appeared heterozygous at the ITS2 locus (i.e., none produced two bands), we inferred that the genetic groups of C. pruni, whose distribution is partly sympatric, constitute biological species that have not exchanged genes for an extended period of time. Other psyllid species (Cacopsylla, Psylla, Triozidae and Aphalaridae) failed to yield any amplicon. These primers are therefore unlikely to produce false positives and allow rapid assignment of C. pruni individuals to either cryptic 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.

Multilocus sequence analysis reveals the genetic diversity of European fruit tree phytoplasmas and supports the existence of inter-species recombination.

The genetic diversity of three temperate fruit tree phytoplasmas 'Candidatus Phytoplasma prunorum', 'Ca. P. mali' and 'Ca. P. pyri' has been established by multilocus sequence analysis. Among the four genetic loci used, the genes imp and aceF distinguished 30 and 24 genotypes, respectively, and showed the highest variability. Percentage of substitution for imp ranged from 50 to 68 % according to species. Percentage of substitution varied between 9 and 12 % for aceF, whereas it was between 5 and 6 % for pnp and secY. In the case of 'Ca P. prunorum' the three most prevalent aceF genotypes were detected in both plants and insect vectors, confirming that the prevalent isolates are propagated by insects. The four isolates known to be hypo-virulent had the same aceF sequence, indicating a possible monophyletic origin. Haplotype network reconstructed by eBURST revealed that among the 34 haplotypes of 'Ca. P. prunorum', the four hypo-virulent isolates also grouped together in the same clade. Genotyping of some Spanish and Azerbaijanese 'Ca. P. pyri' isolates showed that they shared some alleles with 'Ca. P. prunorum', supporting for the first time to our knowledge, the existence of inter-species recombination between these two species.

Specific detection and quantification of the phytopathogenic agent 'Candidatus Phytoplasma prunorum'.

'Candidatus Phytoplasma prunorum' is a wall-less bacterium associated with European stone fruit yellows (ESFY), a severe disease of Prunus spp. (mainly apricot and Japanese plum trees). It can be spread by one insect vector, Cacopsylla pruni, and by the trade of infected material. The availability of PCR-based methods allowing a sensitive and specific detection of 'Ca. P. prunorum' is crucial for this phytoplasma because, at present, it is uncultured and cannot be detected serologically. We developed a PCR test which, in contrast to the existing detection tools, provides a fast, specific and sensitive detection of 'Ca. P. prunorum' in plants and insects. For studies requiring an absolute quantification of the phytoplasma titer, the same primers were used to develop a real-time PCR assay, including a standard for C. pruni. The sensitivity of these molecular tools was compared by serial dilutions and their specificity was assessed both in silico and experimentally for reference strains and field samples of the closely related phytoplasma 'Ca. P. prunorum', 'Ca. P. pyri' (pear decline agent) and 'Ca. P. mali' (apple proliferation agent), as well as for representative strains of the 'Ca. Phytoplasma' genus.

Efficient transmission of 'Candidatus phytoplasma prunorum' Is delayed by eight months due to a long latency in its host-alternating vector.

Understanding at which spatiotemporal scale a disease causes significant secondary spread has both theoretical and practical implications. We investigated this issue in the case of European stone fruit yellows (ESFY), a quarantine vector-borne phytoplasma disease of Prunus trees. Our work was focused on the processes underlying disease spread: the interplay between the life cycles of the pathogen ('Candidatus Phytoplasma prunorum') and of the vector (Cacopsylla pruni). We demonstrated experimentally that C. pruni has only one generation per year and we showed that, at least in southeastern France, C. pruni migrates between conifers in mountainous regions (where it overwinters) and Prunus spp. at lower altitude (where it breeds). In acquisition-inoculation experiments performed with C. pruni over its period of presence on Prunus spp., both immature and mature C. pruni were hardly infectious (0.6%) despite effective phytoplasma acquisition and multiplication. We demonstrated that most immature vectors born on infected plants reach their maximum phytoplasma load (10(7) genomes per insect) only after migrating to conifers and that, after a life-long retention of the phytoplasma, their transmission efficiency was very high (60%) at the end of winter (when they migrate back to their Prunus host). Thus, most transmissions occur only after an effective latency of 8 months, following vector migrations and overwintering on conifers in mountainous regions. From this transmission cycle, we can infer that local secondary spread of ESFY in apricot orchards is marginal, and recommend that disease management strategies take more into account the processes occurring at a regional scale, including the role of wild Prunus spp. in ESFY epidemics.

Nine polymorphic microsatellite loci from the psyllid Cacopsylla pruni (Scopoli), the vector of European stone fruit yellows.

Cacopsylla pruni is the vector of European stone fruit yellows, a quarantine disease of Prunus trees. Nine polymorphic microsatellite markers were developed from enriched DNA libraries. Allelic variability was assessed in a collection of 149 females obtained from five localities covering a large geographical area in France. The number of detected alleles ranged from 8 to 37. Within the localities, observed and expected heterozygosities averaged across loci ranged from 0.39 to 0.55, and from 0.68 to 0.81, respectively. A heterozygote deficiency was detected for almost all loci, possibly due to a high null allele frequency. Other possible causes of the homozygote excess (mode of reproduction, inbreeding, assortative mating or Wahlund effect) are discussed. These variable microsatellite loci can provide tools to assess overall genetic variation in this important vector species. They will be used to search for population structure and migration patterns of C. pruni.

Distinct viral populations differentiate and evolve independently in a single perennial host plant.

The complex structure of virus populations has been the object of intensive study in bacteria, animals, and plants for over a decade. While it is clear that tremendous genetic diversity is rapidly generated during viral replication, the distribution of this diversity within a single host remains an obscure area in this field of science. Among animal viruses, only Human immunodeficiency virus and Hepatitis C virus populations have recently been thoroughly investigated at an intrahost level, where they are structured as metapopulations, demonstrating that the host cannot be considered simply as a "bag" containing a homogeneous or unstructured swarm of mutant viral genomes. In plants, a few reports suggested a possible heterogeneous distribution of virus variants at different locations within the host but provided no clues as to how this heterogeneity is structured. Here, we report the most exhaustive study of the structure and evolution of a virus population ever reported at the intrahost level through the analysis of a Prunus tree infected by Plum pox virus for over 13 years following a single inoculation event and by using analysis of molecular variance at different hierarchical levels combined with nested clade analysis. We demonstrate that, following systemic invasion of the host, the virus population differentiates into several distinct populations that are isolated in different branches, where they evolve independently through contiguous range expansion while colonizing newly formed organs. Moreover, we present and discuss evidence that the tree harbors a huge "bank" of viral clones, each isolated in one of the myriad leaves.

Identifying risk factors for European stone fruit yellows from a survey.

ABSTRACT European stone fruit yellows (ESFY) is becoming a major economic problem for Prunus growers in Europe. The causal agent ("Candidatus Phytoplasma prunorum") and its vector (Cacopsylla pruni) have been identified, but the present knowledge of the risk factors for this disease relies, at best, on specific experiments. To assess the relative significance of several factors correlated with ESFY incidence in the field, an exhaustive survey was performed on apricot and Japanese plum orchards in the Crau plain (France). After a preliminary multivariate exploration of the data, we used a logistic regression model to analyze and predict the cumulative number of diseased trees on the basis of a set of quantitative (age, planting density, and area of the orchard) and categorical variables (species, cultivar, and rootstock). Because of the nature of the data, we used an overdispersed binomial model and we developed a parametric bootstrap procedure based on the beta-binomial distribution to obtain confidence intervals. Our results indicated that the age, species, and cultivar of the scion were the major factors explaining the observed number of diseased trees. The planting density and the rootstocks used in the zone under study were less significant, and the area of the orchard had no effect. The residuals of the model showed that some explanatory variables had not been taken into account, because part of the remaining variability could be explained by a grower effect. The spatial distribution of the residuals suggested that one of the reasons for this grower effect was the correlation between orchards closer than 100 m, possibly caused by the flight behavior of infectious vectors.

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.

Geographically and temporally distant natural recombinant isolates of Plum pox virus (PPV) are genetically very similar and form a unique PPV subgroup.

Natural recombinant Plum pox virus (PPV) isolates were detected in Albania, Bulgaria, Czech Republic, Germany, Hungary and Slovakia. Despite different geographical origins and dates of isolation, all the recombinant isolates were closely related at the molecular level and shared the same recombination breakpoint as well as a typical signature in their N-terminal coat protein sequence, suggesting a common origin. Biological assays with four recombinant isolates demonstrated their capacity to be aphid-transmitted to various Prunus hosts. One of these isolates had a threonine-to-isoleucine mutation in the conserved PTK motif of its HC-Pro and showed a drastically decreased, although not abolished, aphid transmissibility. The complete genome sequence of one of the recombinant isolates, BOR-3, was determined, as well as some partial sequences in the HC-Pro and P3 genes for additional natural recombinant isolates. Analysis of the phylogenetic relationships between the recombinant isolates and other sequenced PPV isolates confirmed that the recombinant isolates form a phylogenetically homogeneous lineage. In addition, this analysis revealed an ancient recombination event between the PPV-D and M subgroups, with a recombination breakpoint located in the P3 gene. Taken together, these results indicate that recombinant isolates represent an evolutionarily successful, homogeneous group of isolates with a common history and unique founding recombination event. The name PPV-Rec is proposed for this coherent ensemble of isolates.

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.

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.