Bayesian inference for spatio-temporal stochastic transmission of plant disease in the presence of roguing: A case study to characterise the dispersal of Flavescence dorée.

Estimating the distance at which pathogens disperse from one season to the next is crucial for designing efficient control strategies for invasive plant pathogens and a major milestone in the reduction of pesticide use in agriculture. However, we still lack such estimates for many diseases, especially for insect-vectored pathogens, such as Flavescence dorée (FD). FD is a quarantine disease threatening European vineyards. Its management is based on mandatory insecticide treatments and the removal of infected plants identified during annual surveys. This paper introduces a general statistical framework to model the epidemiological dynamics of FD in a mechanistic manner that can take into account missing hosts in surveyed fields (resulting from infected plant removals). We parameterized the model using Markov chain Monte Carlo (MCMC) and data augmentation from surveillance data gathered in Bordeaux vineyards. The data mainly consist of two snapshot maps of the infectious status of all the plants in three adjacent fields during two consecutive years. We demonstrate that heavy-tailed dispersal kernels best fit the spread of FD and that on average, 50% (resp. 80%) of new infection occurs within 10.5 m (resp. 22.2 m) of the source plant. These values are in agreement with estimates of the flying capacity of Scaphoideus titanus, the leafhopper vector of FD, reported in the literature using mark-capture techniques. Simulations of simple removal scenarios using the fitted model suggest that cryptic infection hampered FD management. Future efforts should explore whether strategies relying on reactive host removal can improve FD management.

When a Palearctic bacterium meets a Nearctic insect vector: Genetic and ecological insights into the emergence of the grapevine Flavescence dorée epidemics in Europe.

Flavescence dorée (FD) is a European quarantine grapevine disease transmitted by the Deltocephalinae leafhopper Scaphoideus titanus. Whereas this vector had been introduced from North America, the possible European origin of FD phytoplasma needed to be challenged and correlated with ecological and genetic drivers of FD emergence. For that purpose, a survey of genetic diversity of these phytoplasmas in grapevines, S. titanus, black alders, alder leafhoppers and clematis were conducted in five European countries. Out of 132 map genotypes, only 11 were associated to FD outbreaks, three were detected in clematis, whereas 127 were detected in alder trees, alder leafhoppers or in grapevines out of FD outbreaks. Most of the alder trees were found infected, including 8% with FD genotypes M6, M38 and M50, also present in alders neighboring FD-free vineyards and vineyard-free areas. The Macropsinae Oncopsis alni could transmit genotypes unable to achieve transmission by S. titanus, while the Deltocephalinae Allygus spp. and Orientus ishidae transmitted M38 and M50 that proved to be compatible with S. titanus. Variability of vmpA and vmpB adhesin-like genes clearly discriminated 3 genetic clusters. Cluster Vmp-I grouped genotypes only transmitted by O. alni, while clusters Vmp-II and -III grouped genotypes transmitted by Deltocephalinae leafhoppers. Interestingly, adhesin repeated domains evolved independently in cluster Vmp-I, whereas in clusters Vmp-II and-III showed recent duplications. Latex beads coated with various ratio of VmpA of clusters II and I, showed that cluster II VmpA promoted enhanced adhesion to the Deltocephalinae Euscelidius variegatus epithelial cells and were better retained in both E. variegatus and S. titanus midguts. Our data demonstrate that most FD phytoplasmas are endemic to European alders. Their emergence as grapevine epidemic pathogens appeared restricted to some genetic variants pre-existing in alders, whose compatibility to S. titanus correlates with different vmp gene sequences and VmpA binding properties.

Field and Landscape Risk Factors Impacting Flavescence Dorée Infection: Insights from Spatial Bayesian Modeling in the Bordeaux Vineyards.

Flavescence dorée (FD) is a quarantine disease threatening European vineyards. Its management is based on mandatory insecticide treatments and the uprooting of infected plants identified during annual surveys. Field surveys are currently not optimized because the drivers affecting FD spread in vineyard landscapes remain poorly understood. We collated a georeferenced dataset of FD detection, collected from 34,581 vineyard plots over 5 years in the South West France wine region. Spatial models fitted with integrated nested Laplace approximation were used to identify local and landscape factors affecting FD detection and infection. Our analysis highlights the importance of sampling period on FD detection and of local practices and landscape context on FD infection. At field scale, altitude and cultivar choice were the main factors affecting FD infection. In particular, the odds ratio of FD infection in fields planted with the susceptible Cabernet Sauvignon, Cabernet Franc, or Muscadelle varieties were approximately twice those in fields planted with the less susceptible Merlot. Field infection was also affected by the field's immediate surroundings (within a circle with a radius of 150 to 200 m), corresponding to landscapes of 7 to 12 ha. In particular, the probability of FD infection increased with the proportions of forest and urban land and with the proportion of susceptible cultivars, demonstrating that the cultivar composition impacts FD epidemiology at landscape scale. The satisfactory predictive performance of the model for identifying districts with a prevalence of FD detection >10% of the fields suggests that it could be used to target areas in which future surveys would be most valuable.

Genetic Diversity of Flavescence Dorée Phytoplasmas at the Vineyard Scale.

To study the role of wild areas around the vineyards in the epidemiology of flavescence dorée (FD) and track the origin of new foci, two phytoplasma genetic markers, dnaK and malG, were developed for FD phytoplasma (FDp) characterization. The two genes and the vmpA locus were used to genetically characterize FDp populations at seven agroecosystems of a wine-growing Italian region. Vitis vinifera, "gone-wild" V. vinifera and rootstocks, Clematis spp., and Scaphoideus titanus adults were sampled within and outside the vineyards. A range of genotypes infecting the different hosts of the FDp epidemiological cycle was found. Type FD-C isolates were fairly homogeneous compared to type FD-D ones. Most of the FD-D variability was correlated with the malG sequence, and a duplication of this locus was demonstrated for this strain. Coinfection with FD-C and FD-D strains was rare, suggesting possible competition between the two. Similar levels of FDp genetic variation recorded for grapevines or leafhoppers of cultivated and wild areas and co-occurrence of many FDp genotypes inside and outside the vineyards supported the idea of the importance of wild or abandoned Vitis plants and associated S. titanus insects in the epidemiology of the disease. Genetic profiles of FDp found in Clematis were never found in the other hosts, indicating that this species does not take part in the disease cycle in the area. Due to the robustness of analyses using dnaK for discriminating between FD-C and FD-D strains and the high variability of malG sequences, these are efficient markers to study FDp populations and epidemiology at a small geographical scale.IMPORTANCE Flavescence dorée, a threatening disease of grapevine caused by FD phytoplasma (FDp), is distributed within the most important wine-producing areas of Europe and has severe effects on both vineyard productivity and landscape management. FDp is a quarantine pest in Europe, and despite the efforts to contain the pathogen, the disease is still spreading. In this work, new genetic markers for the fine genetic characterization of FDp at local scale are presented. Our findings improve the knowledge of FDp epidemiological cycle and offer the possibility of tracking the route of the FDp infection. In particular, due to its high genetic variability, one of the newly developed markers could be sufficient to track the origin of new infection foci, either from the wild areas or from nurseries.

Variable Membrane Protein A of Flavescence Dorée Phytoplasma Binds the Midgut Perimicrovillar Membrane of Euscelidius variegatus and Promotes Adhesion to Its Epithelial Cells.

Phytoplasmas are uncultivated plant pathogens and cell wall-less bacteria and are transmitted from plant to plant by hemipteran insects. The phytoplasma's circulative propagative cycle in insects requires the crossing of the midgut and salivary glands, and primary adhesion to cells is an initial step toward the invasion process. The flavescence dorée (FD) phytoplasma possesses a set of variable membrane proteins (Vmps) exposed on its surface, and this pathogen is suspected to interact with insect cells. The results showed that VmpA is expressed by the flavescence dorée phytoplasma present in the midgut and salivary glands. Phytoplasmas cannot be cultivated at present, and no mutant can be produced to investigate the putative role of Vmps in the adhesion of phytoplasma to insect cells. To overcome this difficulty, we engineered the Spiroplasma citri mutant G/6, which lacks the ScARP adhesins, for VmpA expression and used VmpA-coated fluorescent beads to determine if VmpA acts as an adhesin in ex vivo adhesion assays and in vivo ingestion assays. VmpA specifically interacted with Euscelidiusvariegatus insect cells in culture and promoted the retention of VmpA-coated beads to the midgut of E. variegatus In this latest case, VmpA-coated fluorescent beads were localized and embedded in the perimicrovillar membrane of the insect midgut. Thus, VmpA functions as an adhesin that could be essential in the colonization of the insect by the FD phytoplasmas.IMPORTANCE Phytoplasmas infect a wide variety of plants, ranging from wild plants to cultivated species, and are transmitted by different leafhoppers, planthoppers, and psyllids. The specificity of the phytoplasma-insect vector interaction has a major impact on the phytoplasma plant host range. As entry into insect cells is an obligate process for phytoplasma transmission, the bacterial adhesion to insect cells is a key step. Thus, studying surface-exposed proteins of phytoplasma will help to identify the adhesins implicated in the specific recognition of insect vectors. In this study, it is shown that the membrane protein VmpA of the flavescence dorée (FD) phytoplasma acts as an adhesin that is able to interact with cells of Euscelidiusvariegatus, the experimental vector of the FD phytoplasma.

Heterologous expression and processing of the flavescence dorée phytoplasma variable membrane protein VmpA in Spiroplasma citri.

BACKGROUND: Flavescence dorée (FD) of grapevine is a phloem bacterial disease that threatens European vineyards. The disease is associated with a non-cultivable mollicute, a phytoplasma that is transmitted by the grapevine leafhopper Scaphoideus titanus in a persistent, propagative manner. The specificity of insect transmission is presumably mediated through interactions between the host tissues and phytoplasma surface proteins comprising the so-called variable membrane proteins (Vmps). Plant spiroplasmas and phytoplasmas share the same ecological niches, the phloem sieve elements of host plants and the hemocoel of insect vectors. Unlike phytoplasmas, however, spiroplasmas, and Spiroplasma citri in particular, can be grown in cell-free media and genetically engineered. As a new approach for studying phytoplasmas-insect cell interactions, we sought to mimic phytoplasmas through the construction of recombinant spiroplasmas exhibiting FD phytoplasma Vmps at the cell surface. RESULTS: Here, we report the expression of the FD phytoplasma VmpA in S. citri. Transformation of S. citri with plasmid vectors in which the vmpA coding sequence was under the control of the S. citri tuf gene promoter resulted in higher accumulation of VmpA than with the native promoter. Expression of VmpA at the spiroplasma surface was achieved by fusing the vmpA coding sequence to the signal peptide sequence of the S. citri adhesin ScARP3d, as revealed by direct colony immunoblotting and immunogold labelling electron microscopy. Anchoring of VmpA to the spiroplasma membrane was further demonstrated by Triton X-114 protein partitioning and Western immunoblotting. Using the same strategy, the secretion of free, functionally active ß-lactamase (used as a model protein) into the culture medium by recombinant spiroplasmas was achieved. CONCLUSIONS: Construction of recombinant spiroplasmas harbouring the FD phytoplasma variable membrane protein VmpA at their surface was achieved, which provides a new biological approach for studying interactions of phytoplasma surface proteins with host cells. Likewise, the secretion of functional ß-lactamase by recombinant spiroplasmas established the considerable promise of the S. citri expression system for delivering phytoplasma effector proteins into host cells.

Flavescence dorée phytoplasma enters insect cells by a clathrin-mediated endocytosis allowing infection of its insect vector.

To perform its propagative and circulative cycle into its insect vector, the flavescence dorée phytoplasma invades different cell types. Clathrin-mediated endocytosis is used by a wide range of bacteria to infect eukaryote cells. Among the insect proteins interacting with the phytoplasma adhesin VmpA, we identified the adaptor protein complex AP-1 and AP-2 suggesting that phytoplasmas could enter the insect cells via clathrin-mediated endocytosis. By infection assays of insect cells in culture, we showed that phytoplasmas entry into Drosophila S2 cells was more efficient than infection of the Euva cell line developed from the insect vector Euscelidius variegatus. Chlorpromazine, cytochalasin D and knockdown of clathrin heavy chain (chc) gene expression using RNA interference inhibited entry of phytoplasmas into S2 cells. During invasion of S2 cells, phytoplasmas were observed very closed to recombinant GFP-labelled clathrin light chain. To verify the role of clathrin in the insect colonization by phytoplasmas, RNAi was performed via artificial feeding of chc dsRNA by the vector E. variegatus. This decreased the expression of chc gene in the midgut and heads of E. variegatus. The chc lower expression correlated to a decreased of midgut and salivary gland cells colonization after the insects had ingested phytoplasmas from infected plants. In conclusion, results indicate that clathrin is important for the FD phytoplasma to enter insect cells and colonize its insect vector.

Epidemiology of flavescence dorée and hazelnut decline in Slovenia: geographical distribution and genetic diversity of the associated 16SrV phytoplasmas.

Flavescence dorée (FD) phytoplasma from 16SrV-C and -D subgroups cause severe damage to grapevines throughout Europe. This phytoplasma is transmitted from grapevine to grapevine by the sap-sucking leafhopper Scaphoideus titanus. European black alder and clematis serve as perennial plant reservoirs for 16SrV-C phytoplasma strains, and their host range has recently been extended to hazelnuts. In Slovenia, hazelnut orchards are declining due to 16SrV phytoplasma infections, where large populations of the non-autochthonous leafhopper Orientus ishidae have been observed. To better characterise the phytoplasma-induced decline of hazelnut and possible transmission fluxes between these orchards and grapevine, genetic diversity of 16SrV phytoplasmas in grapevine, hazelnut and leafhoppers was monitored from 2017 to 2022. The nucleotide sequence analysis was based on the map gene. The most prevalent map genotype in grapevine in all wine-growing regions of Slovenia was M54, which accounted for 84% of the 176 grapevines tested. Besides M54, other epidemic genotypes with lower frequency were M38 (6%), M51 (3%), M50 (2%) and M122 (1%). M38, M50 and M122 were also detected in infected cultivated hazelnuts and in specimens of O. ishidae leafhopper caught in declining hazelnut orchards. It suggests that this polyphagous vector could be responsible for phytoplasma infection in hazelnut orchards and possibly for some phytoplasma exchanges between hazelnuts and grapevine. We hereby describe new genotypes: M158 in grapevine as well as four never reported genotypes M159 to M162 in hazelnut. Of these four genotypes in hazelnut, one (M160) was also detected in O. ishidae. Analysis of additional genes of the new genotypes allowed us to assign them to the VmpA-III cluster, which corresponds to the 16SrV-C strains previously shown to be compatible with S. titanus transmission.

Corrigendum: Epidemiology of flavescence dorée and hazelnut decline in Slovenia: geographical distribution and genetic diversity of the associated 16SrV phytoplasmas.

[This corrects the article DOI: 10.3389/fpls.2023.1217425.].

A knockdown gene approach identifies an insect vector membrane protein with leucin-rich repeats as one of the receptors for the VmpA adhesin of flavescence dorée phytoplasma.

Introduction: The adhesion of flavescence dorée phytoplasma to the midgut epithelium cells of their insect vectors is partially mediated by the variable membrane protein A (VmpA), an adhesin which shows lectin properties. In order to identify the insect receptor for VmpA, we identified Euscelidius variegatus cell proteins interacting with recombinant VmpA-His6. Methods: The E. variegatus proteins were identified by mass spectrometry analysis of VmpA-E. variegatus protein complexes formed upon in vitro interaction assays. To assess their impact in VmpA binding, we reduced the expression of the candidate genes on E. variegatus cells in culture by dsRNA-mediated RNAi. The effect of candidate gene knockdown on VmpA binding was measured by the capacity of E. variegatus cells to bind VmpA-coated fluorescent beads. Results and discussion: There were 13 candidate proteins possessing potential N-glycosylation sites and predicted transmembrane domains selected. The decrease of expression of an unknown transmembrane protein with leucine-rich repeat domains (uk1_LRR) was correlated with the decreased adhesion of VmpA beads to E. variegatus cells. The uk1_LRR was more expressed in digestive tubes than salivary glands of E. variegatus. The protein uk1_LRR could be implicated in the binding with VmpA in the early stages of insect infection following phytoplasmas ingestion.

Genetic diversity of European phytoplasmas of the 16SrV taxonomic group and proposal of 'Candidatus Phytoplasma rubi'.

In addition to the grapevine flavescence dorée phytoplasmas, other members of taxonomic group 16SrV phytoplasmas infect grapevines, alders and species of the genera Clematis and Rubus in Europe. In order to investigate which phytoplasmas constitute discrete, species-level taxa, several strains were analysed by comparing their 16S rRNA gene sequences and a set of five housekeeping genes. Whereas 16S rRNA gene sequence similarity values were >97.5 %, the proposed threshold to distinguish two 'Candidatus Phytoplasma' taxa, phylogenetic analysis of the combined sequences of the tuf, rplV-rpsC, rplF-rplR, map and uvrB-degV genetic loci showed that two discrete phylogenetic clusters could be clearly distinguished. The first cluster grouped flavescence dorée (FD) phytoplasmas, alder yellows (AldY) phytoplasmas, Clematis (CL) phytoplasmas and the Palatinate grapevine yellows (PGY) phytoplasmas. The second cluster comprised Rubus stunt (RS) phytoplasmas. In addition to the specificity of the insect vector, the Rubus stunt phytoplasma contained specific sequences in the 16S rRNA gene. Hence, the Rubus stunt phytoplasma 16S rRNA gene was sufficiently differentiated to represent a novel putative taxon: 'Candidatus Phytoplasma rubi'.

Interactions between the flavescence dorée phytoplasma and its insect vector indicate lectin-type adhesion mediated by the adhesin VmpA.

The flavescence dorée phytoplasma undergoes a propagative cycle in its insect vectors by first interacting with the insect cell surfaces, primarily in the midgut lumen and subsequently in the salivary glands. Adhesion of flavescence dorée phytoplasma to insect cells is mediated by the adhesin VmpA. We hypothesize that VmpA may have lectin-like activity, similar to several adhesins of bacteria that invade the insect gut. We first demonstrated that the luminal surface of the midgut and the basal surface of the salivary gland cells of the natural vector Scaphoideus titanus and those of the experimental vector Euscelidius variegatus were differentially glycosylated. Using ELISA, inhibition and competitive adhesion assays, and protein overlay assays in the Euva-6 insect cell line, we showed that the protein VmpA binds insect proteins in a lectin-like manner. In conclusion, the results of this study indicate that N-acetylglucosamine and mannose present on the surfaces of the midgut and salivary glands serve as recognition sites for the phytoplasma adhesin VmpA.

Contrasting Susceptibilities to Flavescence Dorée in Vitis vinifera, Rootstocks and Wild Vitis Species.

Flavescence dorée (FD) is a quarantine disease of grapevine, involving interactions between the plants, leafhopper vectors, and FD phytoplasma. Characterizing the susceptibility of vine varieties could limit disease propagation. After extensive surveys in vineyards, we showed that Cabernet Sauvignon (CS) is highly susceptible, with a high proportion of symptomatic branches and phytoplasma titers, in contrast to Merlot (M). Localized insect transmissions and grafting showed that phytoplasma circulate in the whole plant in the CS cultivar, but in M they are restricted to the transmission point. Insect-mediated transmission under high confinement mimicking natural conditions confirmed these phenotypes and allowed the classification of 28 Vitis accessions into three distinct categories, according to the percentage of infected plants and their phytoplasma titers. Reduced symptoms, low phytoplasma titers, and low percentages of infected plants were found to be associated in the Vitis vinifera cultivars tested. Interestingly, the low susceptibility of M was observed for one of its parents, i.e., Magdeleine Noire des Charentes. Rootstocks and their Vitis parents, although having high percentages of infected plants and intermediate to high phytoplasma titers, shared a symptomless response. This is troubling, because rootstocks can constitute a silent reservoir of contamination in mother plants or when they grow wild nearby vineyards. Altogether, data suggest distribution of genetic traits within the Vitis genus involved in insect-mediated phytoplasma transmission, multiplication, circulation, and symptom development.

Mapping the phytoplasma chromosome.

Physical and genetic mapping of the phytoplasma chromosome can be a useful tool in a genome sequencing project in order to assemble the in silico-predicted contigs robustly. Mapping consists of four distinct steps: preparation of phytoplasma chromosomes from infected plants, single- and double-digestion of chromosomes with rare-cutting restriction enzymes, separation of large DNA fragments by pulsed-field gel electrophoresis, and hybridization with various genetic markers. Materials and methods needed for each step are described and the technique is illustrated using the flavescence dorée phytoplasma genome map as an example.

Tuf and secY PCR amplification and genotyping of phytoplasmas.

Tuf and secY genotyping techniques have been developed to distinguish phytoplasma strains. Tuf polymerase chain reaction sequence analyses are available for phytoplasma taxonomic groups 16SrI, 16SrV, 16SrXII-A, and XII-B. In addition to their use to confirm the taxonomic status of phytoplasma strains, they allow the spread of phytoplasma strains in host plants and insect vectors to be traced. SecY is more variable than tuf and is therefore more discriminatory than tuf, but secY and tuf phylogenies show congruence.

A chromosome map of the Flavescence doree phytoplasma.

The Flavescence dorée phytoplasma (FD-P), a non-cultivable, plant-pathogenic bacterium of the class Mollicutes, is the causal agent of a quarantine disease affecting vineyards of southern Europe, mainly in southern France and northern Italy. To investigate FD-P diversity and phytoplasma genetic determinants governing the FD-P life cycle, a genome project has been initiated. A physical map of the chromosome of FD-P strain FD92, purified from infected broad beans, was constructed by performing restriction digests of the chromosome and resolving the fragments by PFGE. Single and double digestions of the chromosome with the enzymes SalI, BssHII, MluI and EagI were performed and used to map 13 restriction sites on the FD-P chromosome. The size of the chromosome was calculated to be 671 kbp. Southern blot analyses using cloned phytoplasma probes were carried out to assist in the arrangement of contiguous restriction fragments and to map eight genetic loci, including the two rRNA operons, the tuf, uvrB-degV and secY-map (FD9) genes, the FD2 marker and two orphan sequences (FDDH29 and FDSH05) isolated through subtractive suppression hybridization.

Multilocus sequence typing confirms the close genetic interrelatedness of three distinct flavescence dorée phytoplasma strain clusters and group 16SrV phytoplasmas infecting grapevine and alder in Europe.

Vineyards of southern France and northern Italy are affected by the flavescence dorée (FD) phytoplasma, a quarantine pathogen transmitted by the leafhopper of Nearctic origin Scaphoideus titanus. To better trace propagation of FD strains and identify possible passage between the vineyard and wild plant compartments, molecular typing of phytoplasma strains was applied. The sequences of the two genetic loci map and uvrB-degV, along with the sequence of the secY gene, were determined among a collection of FD and FD-related phytoplasmas infecting grapevine, alder, elm, blackberry, and Spanish broom in Europe. Sequence comparisons and phylogenetic analyses consistently indicated the existence of three FD phytoplasma strain clusters. Strain cluster FD1 (comprising isolate FD70) displayed low variability and represented 17% of the disease cases in the French vineyard, with a higher incidence of the cases in southwestern France. Strain cluster FD2 (comprising isolates FD92 and FD-D) displayed no variability and was detected both in France (83% of the cases) and in Italy, whereas the more-variable strain cluster FD3 (comprising isolate FD-C) was detected only in Italy. The clonal property of FD2 and its wide distribution are consistent with diffusion through propagation of infected-plant material. German Palatinate grapevine yellows phytoplasmas (PGY) appeared variable and were often related to some of the alder phytoplasmas (AldY) detected in Italy and France. Finally, phylogenetic analyses concluded that FD, PGY, and AldY were members of the same phylogenetic subclade, which may have originated in Europe.