Distribution and prevalence of viral genomes in Italian populations of the invasive brown marmorated stink bug Halyomorpha halys.

Halyomorpha halys (Stål), the brown marmorated stink bug, is a highly invasive insect species that causes significant agricultural losses, especially to orchard fruits, vegetables, herbaceous and ornamental plants. It is also a nuisance pest that seeks shelter in indoor spaces during the winter months. Harnessing the H. halys virome can result in new environmentally sustainable approaches to contain its populations and its relatated agricultural damages. In this study, RNA-Seq data were used to explore the virome associated to ten field populations collected in the Lombardy region in Northern Italy. We identified six complete viral genomes, three of which were previously unknown, belonging to the orders Reovirales, Articulavirales, Ghabrivirales, Durnavirales, and Picornavirales. The prevalence of the six viruses was evaluated by Real-time reverse transcription-quantitative PCR on eighty individuals. Halyomorpha halys ifla-like virus 2 turned out to be the most geographically widespread virus, as it was found in more than 50% of the analyzed insects and in nine out of the ten sampling locations. Moreover, in some individuals, this iflavirus was found in association with each of the other viruses in various combinations that involved up to four viruses. Further studies on such virus-virus interactions and their relationships with the insect host may open the possibility to exploit these naturally occurring viruses as specific and targeted biocontrol agents of H. halys.

Silencing of ATP Synthase ß Impairs Egg Development in the Leafhopper Scaphoideus titanus, Vector of the Phytoplasma Associated with Grapevine Flavescence Dorée.

Scaphoideus titanus (Hemiptera: Cicadellidae) is the natural vector of Flavescence dorée phytoplasma, a quarantine pest of grapevine with severe impact on European viticulture. RNA interference (RNAi) machinery components are present in S. titanus transcriptome and injection of ATP synthase ß dsRNAs into adults caused gene silencing, starting three days post injection (dpi) up to 20 dpi, leading to decrease cognate protein. Silencing of this gene in the closely related leafhopper Euscelidiusvariegatus previously showed female sterility and lack of mature eggs in ovaries. Here, alteration of developing egg morphology in S. titanus ovaries as well as overexpression of hexamerin transcript (amino acid storage protein) and cathepsin L protein (lysosome proteinase) were observed in dsATP-injected females. To evaluate RNAi-specificity, E.variegatus was used as dsRNA-receiving model-species. Different doses of two sets of dsRNA-constructs targeting distinct portions of ATP synthase ß gene of both species induced silencing, lack of egg development, and female sterility in E. variegatus, indicating that off-target effects must be evaluated case by case. The effectiveness of RNAi in S. titanus provides a powerful tool for functional genomics of this non-model species and paves the way toward RNAi-based strategies to limit vector population, despite several technical and regulatory constraints that still need to be overcome to allow open field application.

Biological characterization of Euscelidius variegatus iflavirus 1.

Virus-based biocontrol technologies represent sustainable alternatives to pesticides and insecticides. Phytoplasmas are prokaryotic plant pathogens causing severe losses to crops worldwide. Novel approaches are needed since insecticides against their insect vectors and rogueing of infected plants are the only available strategies to counteract phytoplasma diseases. A new iflavirus, named EVV-1, has been described in the leafhopper phytoplasma vector Euscelidius variegatus, raising the potential to use virus-based application strategies against phytoplasma disease. Here transmission routes of EVV-1 are characterized, and localization within the host reveals the mechanism of insect tolerance to virus infection. Both vertical and horizontal transmission of EVV-1 occur and vertical transmission was more efficient. The virus is systemic and occurs in all life-stages, with the highest loads measured in ovaries and first to third instar nymphs. The basic knowledge gained here on the biology of the virus is crucial for possible future application of iflaviruses as biocontrol agents.

Two Phytoplasmas Elicit Different Responses in the Insect Vector Euscelidius variegatus Kirschbaum.

Phytoplasmas are plant-pathogenic bacteria transmitted by hemipteran insects. The leafhopper Euscelidius variegatus is a natural vector of chrysanthemum yellows phytoplasma (CYp) and a laboratory vector of flavescence dorée phytoplasma (FDp). The two phytoplasmas induce different effects on this species: CYp slightly improves whereas FDp negatively affects insect fitness. To investigate the molecular bases of these different responses, transcriptome sequencing (RNA-seq) analysis of E. variegatus infected with either CYp or FDp was performed. The sequencing provided the first de novo transcriptome assembly for a phytoplasma vector and a starting point for further analyses on differentially regulated genes, mainly related to immune system and energy metabolism. Insect phenoloxidase activity, immunocompetence, and body pigmentation were measured to investigate the immune response, while respiration and movement rates were quantified to confirm the effects on energy metabolism. The activation of the insect immune response upon infection with FDp, which is not naturally transmitted by E. variegatus, confirmed that this bacterium is mostly perceived as a potential pathogen. Conversely, the acquisition of CYp, which is naturally transmitted by E. variegatus, seems to increase the insect fitness by inducing a prompt response to stress. This long-term relationship is likely to improve survival and dispersal of the infected insect, thus enhancing the opportunity of phytoplasma transmission.

Genome sequence, prevalence and quantification of the first iflavirus identified in a phytoplasma insect vector.

The leafhopper Euscelidius variegatus is a natural vector of chrysanthemum yellows phytoplasma (CY) and an efficient vector of flavescence dorée phytoplasma (FD) under laboratory conditions. During a transcriptome sequencing (RNA-seq) project aimed at investigating the interactions between the insect and the two phytoplasmas, a 10,616-nucleotide-long contig with high sequence similarity to known picorna-like viruses was identified among the assembled insect transcripts. The discovery came totally unexpected, because insects from the laboratory colony did not show any evident symptom that could be related to the presence of a virus. The amino acid sequence, the shape and size of viral particles, and the results of phylogenetic analysis suggest that this virus, named Euscelidius variegatus virus 1 (EVV-1), can be considered a new member of a new species in the genus Iflavirus. EVV-1 was detected in all of the tested insects from the laboratory colony used for RNA-seq, both in phytoplasma-exposed and in non-exposed insects, but the viral load measured in FD-exposed samples was significantly lower than that in non-exposed insects. This result suggests the possible existence of an intriguing cross-talk among insects, endogenous bacteria, and viruses. The identification of two other E. variegatus laboratory colonies that were free of EVV-1 could represent the key to addressing some basic virological issues, e.g., viral replication and transmission mechanisms, and offer the opportunity to use infectious clones to express heterologous genes in the leafhopper and manipulate the expression of endogenous genes by promoting virus-induced gene silencing.

Acquisition of Flavescence Dorée Phytoplasma by Scaphoideus titanus Ball from Different Grapevine Varieties.

Flavescence dorée (FD) is a threat for wine production in the vineyard landscape of Piemonte, Langhe-Roero and Monferrato, Italy. Spread of the disease is dependent on complex interactions between insect, plant and phytoplasma. In the Piemonte region, wine production is based on local cultivars. The role of six local grapevine varieties as a source of inoculum for the vector Scaphoideus titanus was investigated. FD phytoplasma (FDP) load was compared among red and white varieties with different susceptibility to FD. Laboratory-reared healthy S. titanus nymphs were caged for acquisition on infected plants to measure phytoplasma acquisition efficiency following feeding on different cultivars. FDP load for Arneis was significantly lower than for other varieties. Acquisition efficiency depended on grapevine variety and on FDP load in the source plants, and there was a positive interaction for acquisition between variety and phytoplasma load. S. titanus acquired FDP with high efficiency from the most susceptible varieties, suggesting that disease diffusion correlates more with vector acquisition efficiency than with FDP load in source grapevines. In conclusion, although acquisition efficiency depends on grapevine variety and on FDP load in the plant, even varieties supporting low FDP multiplication can be highly susceptible and good sources for vector infection, while poorly susceptible varieties may host high phytoplasma loads.

Role of the major antigenic membrane protein in phytoplasma transmission by two insect vector species.

BACKGROUND: Phytoplasmas are bacterial plant pathogens (class Mollicutes), transmitted by phloem feeding leafhoppers, planthoppers and psyllids in a persistent/propagative manner. Transmission of phytoplasmas is under the control of behavioral, environmental and geographical factors, but molecular interactions between membrane proteins of phytoplasma and vectors may also be involved. The aim of the work was to provide experimental evidence that in vivo interaction between phytoplasma antigenic membrane protein (Amp) and vector proteins has a role in the transmission process. In doing so, we also investigated the topology of the interaction at the gut epithelium and at the salivary glands, the two barriers encountered by the phytoplasma during vector colonization. METHODS: Experiments were performed on the 'Candidatus Phytoplasma asteris' chrysanthemum yellows strain (CYP), and the two leafhopper vectors Macrosteles quadripunctulatus Kirschbaum and Euscelidius variegatus Kirschbaum. To specifically address the interaction of CYP Amp at the gut epithelium barrier, insects were artificially fed with media containing either the recombinant phytoplasma protein Amp, or the antibody (A416) or both, and transmission, acquisition and inoculation efficiencies were measured. An abdominal microinjection protocol was employed to specifically address the interaction of CYP Amp at the salivary gland barrier. Phytoplasma suspension was added with Amp or A416 or both, injected into healthy E. variegatus adults and then infection and inoculation efficiencies were measured. An internalization assay was developed, consisting of dissected salivary glands from healthy E. variegatus exposed to phytoplasma suspension alone or together with A416 antibody. The organs were then either observed in confocal microscopy or subjected to DNA extraction and phytoplasma quantification by qPCR, to visualize and quantify possible differences among treatments in localization/presence/number of CYP cells. RESULTS: Artificial feeding and abdominal microinjection protocols were developed to address the two barriers separately. The in vivo interactions between Amp of 'Candidatus Phytoplasma asteris' Chrysanthemum yellows strain (CYP) and vector proteins were studied by evaluating their effects on phytoplasma transmission by Euscelidius variegatus and Macrosteles quadripunctulatus leafhoppers. An internalization assay was developed, consisting of dissected salivary glands from healthy E. variegatus exposed to phytoplasma suspension alone or together with anti-Amp antibody. To visualize possible differences among treatments in localization/presence of CYP cells, the organs were observed in confocal microscopy. Pre-feeding of E. variegatus and M. quadripunctulatus on anti-Amp antibody resulted in a significant decrease of acquisition efficiencies in both species. Inoculation efficiency of microinjected E. variegatus with CYP suspension and anti-Amp antibody was significantly reduced compared to that of the control with phytoplasma suspension only. The possibility that this was due to reduced infection efficiency or antibody-mediated inhibition of phytoplasma multiplication was ruled out. These results provided the first indirect proof of the role of Amp in the transmission process. CONCLUSION: Protocols were developed to assess the in vivo role of the phytoplasma native major antigenic membrane protein in two phases of the vector transmission process: movement through the midgut epithelium and colonization of the salivary glands. These methods will be useful also to characterize other phytoplasma-vector combinations. Results indicated for the first time that native CYP Amp is involved in vivo in specific crossing of the gut epithelium and salivary gland colonization during early phases of vector infection.

RNA-Seq profile of flavescence dorée phytoplasma in grapevine.

BACKGROUND: The phytoplasma-borne disease flavescence dorée is still a threat to European viticulture, despite mandatory control measures and prophylaxis against the leafhopper vector. Given the economic importance of grapevine, it is essential to find alternative strategies to contain the spread, in order to possibly reduce the current use of harmful insecticides. Further studies of the pathogen, the vector and the mechanisms of phytoplasma-host interactions could improve our understanding of the disease. In this work, RNA-Seq technology followed by three de novo assembly strategies was used to provide the first comprehensive transcriptomics landscape of flavescence dorée phytoplasma (FD) infecting field-grown Vitis vinifera leaves. RESULTS: With an average of 8300 FD-mapped reads per library, we assembled 347 sequences, corresponding to 215 annotated genes, and identified 10 previously unannotated genes, 15 polycistronic transcripts and three genes supposedly localized in the gaps of the FD92 draft genome. Furthermore, we improved the annotation of 44 genes with the addition of 5'/3' untranslated regions. Functional classification revealed that the most expressed genes were either related to translation and protein biosynthesis or hypothetical proteins with unknown function. Some of these hypothetical proteins were predicted to be secreted, so they could be bacterial effectors with a potential role in modulating the interaction with the host plant. Interestingly, qRT-PCR validation of the RNA-Seq expression values confirmed that a group II intron represented the FD genomic region with the highest expression during grapevine infection. This mobile element may contribute to the genomic plasticity that is necessary for the phytoplasma to increase its fitness and endorse host-adaptive strategies. CONCLUSIONS: The RNA-Seq technology was successfully applied for the first time to analyse the FD global transcriptome profile during grapevine infection. Our results provided new insights into the transcriptional organization and gene structure of FD. This may represent the starting point for the application of high-throughput sequencing technologies to study differential expression in FD and in other phytoplasmas with an unprecedented resolution.

Competition among Flavescence Dorée Phytoplasma Strains in the Experimental Insect Vector Euscelidius variegatus.

Phytoplasmas are plant pathogenic wall-less bacteria transmitted in a persistent propagative manner by hemipteran insects, mainly belonging to the suborder Auchenorrhyncha (Fulgoromorpha and Cicadomorpha). Flavescence dorée (FD) is a quarantine disease of grapevine, causing great damage to European viticulture and associated with phytoplasmas belonging to 16SrV-C (FD-C) and -D (FD-D) subgroups. FD-C and FD-D strains share similar pathogenicity, but mixed infections are rare in nature. To investigate the competition among FDp strains, specimens of the laboratory vector Euscelidius variegatus (Hemiptera: Cicadellidae) were forced to acquire both phytoplasma haplotypes upon feeding on FD-C- and FD-D-infected plants or after the injection of both strains. The pathogen colonization of insect bodies and heads was monitored with multiplex qPCR, and the efficiencies of phytoplasma transmission were estimated. Single infection, irrespective of strain type, was more frequent than expected, indicating that competition among FD strains occurs. Hypotheses of competition for resources and/or host active sites or the direct antibiosis of one strain against the other are discussed, based on the genetic complexity of FDp populations and on the high genome variability of the FD-D strain. As FD management still mainly relies on insecticides against vectors, the characterization of FDp haplotypes and the description of their epidemiology also have practical implications.

Metatranscriptomic Assessment of the Microbial Community Associated With the Flavescence dorée Phytoplasma Insect Vector Scaphoideus titanus.

Phytoplasmas are insect-borne pathogenic bacteria that cause major economic losses to several crops worldwide. The dynamic microbial community associated with insect vectors influences several aspects of their biology, including their vector competence for pathogens. Unraveling the diversity of the microbiome of phytoplasma insect vectors is gaining increasing importance in the quest to develop novel microbe-based pest control strategies that can minimize the use of insecticides for better environmental quality. The leafhopper Scaphoideus titanus is the primary vector of the Flavescence dorée phytoplasma, a quarantine pest which is dramatically affecting the main grape-growing European countries. In this study, the RNA-Seq data, which were previously used for insect virus discovery, were further explored to assess the composition of the whole microbial community associated with insects caught in the wild in both its native (the United States) and invasive (Europe) areas. The first de novo assembly of the insect transcriptome was used to filter the host sequencing reads. The remaining ones were assembled into contigs and analyzed by blastx to provide the taxonomic identification of the microorganisms associated with S. titanus, including the non-bacterial components. By comparing the transcriptomic libraries, we could differentiate the stable and consistent associations from the more ephemeral and flexible ones. Two species appeared to be universal to the core microbiome of S. titanus: the obligate bacterial symbiont Candidatus Sulcia muelleri and an Ophiocordyceps-allied fungus distantly related to yeast-like symbionts described from other hemipterans. Bacteria of the genus Cardinium have been identified as another dominant member of the microbiome, but only in the European specimens. Although we are yet to witness how the interplay among the microorganisms influences the vector competence of S. titanus, this unbiased in silico characterization of its microbiome is paramount for identifying the naturally occurring targets for new biocontrol strategies to counteract Flavescence dorée spread in Europe.

Silencing of ATP synthase ß reduces phytoplasma multiplication in a leafhopper vector.

The leafhopper Euscelidius variegatus is a natural vector of the chrysanthemum yellows phytoplasma (CYp) and a laboratory vector of the Flavescence dorée phytoplasma (FDp). Previous studies indicated a crucial role for insect ATP synthase α and ß subunits during phytoplasma infection of the vector species. Gene silencing of ATP synthase ß was obtained by injection of specific dsRNAs in E. variegatus. Here we present the long-lasting nature of such silencing, its effects on the small RNA profile, the significant reduction of the corresponding protein expression, and the impact on phytoplasma acquisition capability. The specific transcript expression was silenced at least up to 37 days post injection with an average reduction of 100 times in insects injected with dsRNAs targeting ATP synthase ß (dsATP) compared with those injected with dsRNAs targeting green fluorescent protein (dsGFP), used as negative controls. Specific silencing of this gene was also confirmed at protein level at 15 days after the injection. Total sRNA reads mapping to dsATP and dsGFP sequences in analysed libraries showed in both cases a peak of 21 nt, a length consistent with the generation of dsRNA-derived siRNAs by RNAi pathway. Reads mapped exclusively to the fragment corresponding to the injected dsATPs, probably indicating the absence of a secondary machinery for siRNA synthesis. Insects injected either with dsATP or dsGFP successfully acquired CYp and FDp during feeding on infected plants. However, the average phytoplasma amount in dsATP insects was significantly lower than that measured in dsGFP specimens, indicating a probable reduction of the pathogen multiplication when ATP synthase ß was silenced. The role of the insect ATP synthase ß during phytoplasma infection process is discussed.

Bacteriophage-Host Association in the Phytoplasma Insect Vector Euscelidius variegatus.

Insect vectors transmit viruses and bacteria that can cause severe diseases in plants and economic losses due to a decrease in crop production. Insect vectors, like all other organisms, are colonized by a community of various microorganisms, which can influence their physiology, ecology, evolution, and also their competence as vectors. The important ecological meaning of bacteriophages in various ecosystems and their role in microbial communities has emerged in the past decade. However, only a few phages have been described so far in insect microbiomes. The leafhopper Euscelidius variegatus is a laboratory vector of the phytoplasma causing Flavescence dorée, a severe grapevine disease that threatens viticulture in Europe. Here, the presence of a temperate bacteriophage in E. variegatus (named Euscelidius variegatus phage 1, EVP-1) was revealed through both insect transcriptome analyses and electron microscopic observations. The bacterial host was isolated in axenic culture and identified as the bacterial endosymbiont of E. variegatus (BEV), recently assigned to the genus Candidatus Symbiopectobacterium. BEV harbors multiple prophages that become active in culture, suggesting that different environments can trigger different mechanisms, finely regulating the interactions among phages. Understanding the complex relationships within insect vector microbiomes may help in revealing possible microbe influences on pathogen transmission, and it is a crucial step toward innovative sustainable strategies for disease management in agriculture.

Competitive Exclusion of Flavescence dorée Phytoplasma Strains in Catharanthus roseus Plants.

Flavescence dorée phytoplasmas (FDp, 16SrV-C and -D) are plant pathogenic non-cultivable bacteria associated with a severe grapevine disease. The incidence of the two reference strains on cultivated grapevines is unbalanced, and mixed infections are rare. To investigate the interaction between the two strains, Catharanthus roseus plants were graft-infected with both strains, either simultaneously or sequentially. Different combinations of lateral and apical grafting were applied to avoid possible benefits due to graft position. The infection was monitored for four months through a new diagnostic protocol developed for differentiation and relative quantification of the two strains. Regardless of the temporal or spatial advantage at grafting, FD-C generally outcompeted FD-D. The prevalence of FD-C increased over time and, at the end of the experiment, FD-C was the unique strain detected in the aerial part and the roots of 74% and 90% of grafted plants, respectively. These data indicate that the interaction between the two strains results in competitive exclusion. Understanding the bases of the competition between FD-C and FD-D may contribute to explain the biology of the coexistence of different FDp strains under field conditions, aiming at identifying potential suppressor strains, which can provide alternative and environmentally sustainable solutions for FD control.

New Viral Sequences Identified in the Flavescence Dorée Phytoplasma Vector Scaphoideus titanus.

The leafhopper Scaphoideus titanus is the primary vector of Flavescence dorée phytoplasma (FDp) in European vineyards. Flavescence dorée is one of the most severely damaging diseases of Vitis vinifera and, consequently, a major threat to grape and wine production in several European countries. Control measures are compulsory, but they mainly involve large-scale insecticide treatments, with detrimental impacts on the environment. One possible solution is to exploit the largely unexplored genetic diversity of viruses infecting S. titanus as highly specific and environmentally benign tools for biological control. (2)Methods: A metatranscriptomic approach was adopted to identify viruses that may infect individuals caught in the wild in both its native (United States) and invasive (Europe) areas. Reverse transcription PCR was used to confirm their presence in RNA pools and explore their prevalence. (3)Results: We described nine new RNA viruses, including members of "Picorna-Calici", "Permutotetra", "Bunya-Arena", "Reo", "Partiti-Picobirna", "Luteo-Sobemo" and "Toti-Chryso" clades. A marked difference in the diversity and abundance of the viral species was observed between the US population and the European ones. (4)Conclusions: This work represents the first survey to assess the viral community of a phytoplasma insect vector. The possibility to exploit these naturally occurring viruses as specific and targeted biocontrol agents of S. titanus could be the answer to increasing demand for a more sustainable viticulture.

Pest categorisation of the non-EU phytoplasmas of Cydonia Mill., Fragaria L., Malus Mill., Prunus L., Pyrus L., Ribes L., Rubus L. and Vitis L.

Following a request from the European Commission, the EFSA Panel on Plant Health performed a pest categorisation of nine phytoplasmas of Cydonia Mill., Fragaria L., Malus Mill., Prunus L., Pyrus L., Ribes L., Rubus L. and Vitis L. (hereafter "host plants") known to occur only outside the EU or having a limited presence in the EU. This opinion covers the (i) reference strains of 'Candidatus Phytoplasma australiense', 'Ca. P. fraxini', 'Ca. P. hispanicum', 'Ca. P. trifolii', 'Ca. P. ziziphi', (ii) related strains infecting the host plants of 'Ca. P. aurantifolia', 'Ca. P. pruni', and 'Ca. P. pyri', and (iii) an unclassified phytoplasma causing Buckland valley grapevine yellows. Phytoplasmas can be detected by available methods and are efficiently transmitted by vegetative propagation, with plants for planting acting as a major entry pathway and a long-distance spread mechanism. Phytoplasmas are also transmitted in a persistent and propagative manner by some insect families of the Fulgoromorpha, Cicadomorpha and Sternorrhyncha (order Hemiptera). No transovarial, pollen or seed transmission has been reported. The natural host range of the categorised phytoplasmas varies from one to more than 90 plant species, thus increasing the possible entry pathways. The host plants are widely cultivated in the EU. All the categorised phytoplasmas can enter and spread through the trade of host plants for planting, and by vectors. Establishment of these phytoplasmas is not expected to be limited by EU environmental conditions. The introduction of these phytoplasmas in the EU would have an economic impact. There are measures to reduce the risk of entry, establishment, spread and impact. Uncertainties result from limited information on distribution, biology and epidemiology. All the phytoplasmas categorised here meet the criteria evaluated by EFSA to qualify as potential Union quarantine pests, and they do not qualify as potential regulated non-quarantine pests, because they are non-EU phytoplasmas.

List of non-EU phytoplasmas of Cydonia Mill., Fragaria L., Malus Mill., Prunus L., Pyrus L., Ribes L., Rubus L. and Vitis L.

Following a request from the European Commission, the EFSA Panel on Plant Health prepared a list of non-EU phytoplasmas of Cydonia Mill., Fragaria L., Malus Mill., Prunus L., Pyrus L., Ribes L., Rubus L. and Vitis L. A systematic literature review and search of databases identified 27 phytoplasmas infecting one or more of the host genera under consideration. These phytoplasmas were assigned to three categories. The first group (a) consists of 10 non-EU phytoplasmas, known to occur only outside the EU ('Candidatus Phytoplasma australiense', 'Ca. P. hispanicum', 'Ca. P. pruni'-related strain (NAGYIII), 'Ca. P. pyri'-related strain (PYLR) and Buckland valley grapevine yellows phytoplasma) or having only limited presence in the EU ('Ca. P. aurantifolia'-related strains, 'Ca. P. fraxini', 'Ca. P. phoenicium', 'Ca. P. trifolii' and 'Ca. P. ziziphi'). The second group (b) consists of three non-EU phytoplasmas, whose presence in the target plant species is not fully supported by the available literature. The third group (c) consists of 14 phytoplasmas with substantial presence in the EU (i.e. they are originally described or reported from the EU or known to occur or be widespread in some EU Member States or frequently reported in the EU). Phytoplasmas of categories (b) and (c) were excluded at this stage from further categorisation efforts. One phytoplasma from category (a) ('Ca. P. phoenicium') was excluded from further categorisation, as a pest risk assessment has been performed by EPPO. Comments provided by the EU Member States were integrated in the opinion. The main uncertainties of this listing concern: the geographic distribution and prevalence, the taxonomy, biology and host range. The phytoplasmas considered as non-EU and whose presence in target plant species is fully supported by literature (category (a)) are categorised by the Panel in a separate opinion.

List of non-EU phytoplasmas of tuber-forming Solanum spp.

Following a request from the European Commission, the EFSA Panel on Plant Health prepared a list of non-EU phytoplasmas of tuber-forming Solanum spp. A systematic literature review and search of databases identified 12 phytoplasmas infecting S. tuberosum. These phytoplasmas were assigned to three categories. The first group (a) consists of seven non-EU phytoplasmas, known to occur only outside the EU ('Candidatus Phytoplasma americanum', 'Ca. P. australiense', 'Ca. P. fragariae'-related strain (YN-169, YN-10G) and 'Ca. P. hispanicum') or having only limited presence in the EU ('Ca. P. aurantifolia'-related strains, 'Ca. P. pruni'-related strains and 'Ca. P. trifolii'). The second group (b) consists of three phytoplasmas originally described or reported from the EU. The third group (c) consists of two phytoplasmas with substantial presence in the EU, whose presence in S. tuberosum is not fully supported by the available literature. Phytoplasmas of categories (b) and (c) were excluded at this stage from further categorisation efforts. Three phytoplasmas from category (a) ('Ca. P. australiense', 'Ca. P. hispanicum' and 'Ca. P. trifolii') were excluded from further categorisation, as a pest categorisation has already been performed by EFSA. Comments provided by the EU Member States were integrated in the opinion. The main uncertainties of this listing concern: the taxonomy, the geographic distribution and prevalence and host range. The following phytoplasmas considered as non-EU and whose presence in S. tuberosum is fully supported by literature (category (a)) are categorised by the Panel in a separate opinion: 'Ca. P. americanum', 'Ca. P. fragariae'-related strain (YN-169, YN-10G), 'Ca. P. aurantifolia'-related strains and 'Ca. P. pruni'-related strains.

Pest categorisation of the non-EU phytoplasmas of tuber-forming Solanum spp.

Following a request from the European Commission, the EFSA Panel on Plant Health performed a pest categorisation of four phytoplasmas of tuber-forming Solanum spp. known to occur only outside the EU or having a limited presence in the EU. The only tuber-forming species of Solanum reported to be phytoplasma infected is S. tuberosum. This opinion covers 'Candidatus Phytoplasma americanum', 'Ca. P. aurantifolia'-related strains (GD32; St_JO_10, 14, 17; PPT-SA; Rus-343F; PPT-GTO29, -GTO30, -SINTV; Potato Huayao Survey 2; Potato hair sprouts), 'Ca. P. fragariae'-related strains (YN-169, YN-10G) and 'Ca. P. pruni'-related strains (Clover yellow edge; Potato purple top AKpot7, MT117, AKpot6; PPT-COAHP, -GTOP). Phytoplasmas can be detected by molecular methods and are efficiently transmitted by vegetative propagation. Phytoplasmas are also transmitted in a persistent and propagative manner by some insects belonging to families within Cicadomorpha, Fulgoromorpha and Sternorrhyncha (order Hemiptera). No transovarial, pollen or seed transmission has been reported. The reported natural host range of the phytoplasmas categorised here varies from restricted ('Ca. P. americanum', and 'Ca. P. fragariae'-related strains) to wide ('Ca. P. aurantifolia'-related strains and 'Ca. P. pruni'-related strains), thus increasing the possible entry pathways in the latter case. S. tuberosum is widely cultivated in the EU. All the categorised phytoplasmas can enter and spread through the trade of host plants for planting, and by vectors. Establishment of these phytoplasmas is not expected to be limited by EU environmental conditions. The introduction of these phytoplasmas in the EU would have an economic impact. There are measures to reduce the risk of entry, establishment, spread and impact. Uncertainties result from limited information on distribution, biology and epidemiology. All the phytoplasmas categorised here meet the criteria evaluated by EFSA to qualify as potential Union quarantine pests, and they do not meet all the criteria to qualify as potential regulated non-quarantine pests, because they do not occur or are not known to be widespread in the EU.

Immunofluorescence Assay to Study Early Events of Vector Salivary Gland Colonization by Phytoplasmas.

To visualize phytoplasmas at early stages of vector infection, an immunofluorescence assay was developed. The chapter provides experimental details on dissection of salivary glands, incubation of the dissected organs with phytoplasma suspension, fixation, embedding, sectioning, labeling, and final visualization with confocal microscopy. All the procedure will be described for the leafhopper Euscelidius variegatus, natural vector of "Candidatus phytoplasma asteris" and laboratory vector of Flavescence dorée phytoplasma.

RNA interference of muscle actin and ATP synthase beta increases mortality of the phytoplasma vector Euscelidius variegatus.

BACKGROUND: RNA interference (RNAi) techniques have emerged as powerful tools to develop novel management strategies for the control of insect pests. The leafhopper Euscelidius variegatus is a natural vector of chrysanthemum yellows phytoplasma and a laboratory vector of Flavescence dorée phytoplasma. Phytoplasmas are insect-borne bacterial plant pathogens that cause economically relevant crop losses worldwide. RESULTS: In this study, we demonstrated that microinjection of muscle actin and ATP synthase ß double-stranded (ds)RNAs into adult insects caused an exponential reduction in the expression of both genes, which began within 72 h of dsRNA administration and lasted for 14 days, leading to almost complete silencing of the target genes. Such silencing effects on muscle actin expression appeared to be both time- and dose-dependent. Our results also showed that the knockdown of both genes caused a significant decrease in survival rates in comparison with green fluorescent protein (GFP) dsRNA-injected control insects. CONCLUSION: The effectiveness of RNAi-based gene silencing in E. variegatus guarantees the availability of a powerful reverse genetic tool for the functional annotation of its genes and the identification of those potentially involved in the interaction with phytoplasmas. In addition, this study demonstrated that muscle actin and ATP synthase ß may represent candidate genes for RNAi-based control of E. variegatus. © 2018 Society of Chemical Industry.