Commodity risk assessment of plants of 12 selected Prunus species from Moldova.

The European Commission requested the EFSA Panel on Plant Health to prepare and deliver risk assessments for commodities listed in Commission Implementing Regulation (EU) 2018/2019 as 'High-risk plants, plant products and other objects'. This Scientific Opinion covers plant health risks posed by defoliated 1- or 2-year old bare root plants for planting (grafted or not) of 12 Prunus species (Prunus armeniaca, P. avium, P. canescens, P. cerasifera, P. cerasus, P. davidiana, P. domestica, P. dulcis, P. fontanesiana, P. persica, P. salicina, P. tomentosa) imported from Moldova, taking into account the available scientific information, including the technical information provided by the applicant country. The evaluation identified three EU-quarantine pests, Erwinia amylovora (protected zone quarantine pest), Xiphinema rivesi non-EU populations and Xanthomonas arboricola pv. pruni (protected zone quarantine pest), which were selected for further evaluation, based on defined criteria, including their presence in the applicant country. It should be noted that there is uncertainty regarding whether all relevant pests have been identified due to a limited number of scientific publications and pest surveys in Moldova. For the three selected pests, the risk mitigation measures proposed in the technical dossier from Moldova were evaluated taking into account the possible limiting factors. For these pests, an expert judgement is given on the likelihood of pest freedom taking into consideration the risk mitigation measures acting on it, including uncertainties associated with the assessment. The degree of pest freedom varies among the pests evaluated, with Erwinia amylovora being the pest most frequently expected on the imported plants. The Expert Knowledge Elicitation indicated, with 95% certainty, that between 9823 and 10,000 bundles (comprising 10-20 plants per bundle) out of 10,000 bundles would be free from E. amylovora.

Commodity risk assessment of Malus domestica plants from Bosnia and Herzegovina.

The European Commission requested the EFSA Panel on Plant Health to prepare and deliver risk assessments for commodities listed in Commission Implementing Regulation (EU) 2018/2019 as 'High-risk plants, plant products and other objects'. This Scientific Opinion covers plant health risks posed by bare root plants of Malus domestica grafted on rootstocks of either Malus domestica or Malus sylvestris imported from Bosnia and Herzegovina (BIH), taking into account the available scientific information, including the technical information provided by Bosnia and Herzegovina. All pests associated with the commodities were evaluated against specific criteria for their relevance for this opinion. One protected zone quarantine pest, Erwinia amylovora, fulfilled all relevant criteria and was selected for further evaluation. For E. amylovora, special requirements are specified in Commission Implementing Regulation (EU) 2019/2072. Based on the information provided in the Dossier, these specific requirements for E. amylovora are not met.

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.

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.

Commodity risk assessment of Malus sylvestris plants from United Kingdom.

The European Commission requested the EFSA Panel on Plant Health to prepare and deliver risk assessments for commodities listed in Commission Implementing Regulation (EU) 2018/2019 as 'High risk plants, plant products and other objects'. This Scientific Opinion covers plant health risks posed by rooted plants and bundles of bare root plants or rooted cell grown young plants of Malus sylvestris imported from the UK, taking into account the available scientific information, including the technical information provided by the UK. All pests associated with the commodities were evaluated against specific criteria for their relevance for this opinion. Two quarantine pests (tobacco ringspot virus and tomato ringspot virus), one protected zone quarantine pest (Erwinia amylovora) and four non-regulated pests (Colletotrichum aenigma, Meloidogyne mali, Eulecanium excrescens and Takahashia japonica) that fulfilled all relevant criteria were selected for further evaluation. For Erwinia amylovora, special requirements are specified in Commission Implementing Regulation (EU) 2019/2072. Based on the information provided in the dossier, these specific requirements for E. amylovora are met. For the remaining six pests, the risk mitigation measures proposed in the technical Dossier from the UK were evaluated, taking into account the possible limiting factors. For these pests, expert judgement is given on the likelihood of pest freedom, taking into consideration the risk mitigation measures acting on the pest, including uncertainties associated with the assessment. The degree of pest freedom varies among the pests evaluated, with scales (Eulecanium excrescens and Takahashia japonica) being the pests most frequently expected on the imported bundles of bare root plants or rooted cell grown young plants. The expert knowledge elicitation indicated with 95% certainty that between 9,976 and 10,000 bundles (one bundle consisting of 5-15 plants for bare root plants or 25-50 plants for cell grown young plants) per 10,000 would be free from the above-mentioned scales.

Commodity risk assessment of Malus domestica plants from United Kingdom.

The European Commission requested the EFSA Panel on Plant Health to prepare and deliver risk assessments for commodities listed in Commission Implementing Regulation (EU) 2018/2019 as 'High risk plants, plant products and other objects'. This Scientific Opinion covers plant health risks posed by rooted plants in pots, bundles of bare-rooted plants or trees and bundles of budwood and graftwood of Malus domestica imported from the United Kingdom, taking into account the available scientific information, including the technical information provided by the United Kingdom. All pests associated with the commodities were evaluated against specific criteria for their relevance for this opinion. Two quarantine pests (tobacco ringspot virus and tomato ringspot virus), one protected zone quarantine pest (Erwinia amylovora) and four non-regulated pests (Colletotrichum aenigma, Meloidogyne mali, Eulecanium excrescens, Takahashia japonica) that fulfilled all relevant criteria were selected for further evaluation. For E. amylovora, special requirements are specified in Commission Implementing Regulation (EU) 2019/2072. Based on the information provided in the Dossier, the specific requirements for E. amylovora were met. For the remaining six pests, the risk mitigation measures proposed in the technical Dossier from the UK were evaluated considering the possible limiting factors. For the selected pests, expert judgement is given on the likelihood of pest freedom, taking into consideration the risk mitigation measures acting on the pest, including uncertainties associated with the assessment. The degree of pest freedom varies among the pests evaluated, with scales (E. excrescens and T. japonica) being the pests most frequently expected on the imported budwood and graftwood. The Expert Knowledge Elicitation indicated with 95% certainty that between 9,976 and 10,000 bundles (consisting of 50 up to 500 plants each) per 10,000 would be free from the above-mentioned scales.

Commodity risk assessment of Prunus persica and P. dulcis plants from Türkiye.

The European Commission requested the EFSA Panel on Plant Health to prepare and deliver risk assessments for commodities listed in Commission Implementing Regulation (EU) 2018/2019 as 'High risk plants, plant products and other objects'. This Scientific Opinion covers plant health risks posed by plants of Prunus persica and P. dulcis, as budwood/graftwood, rooted or grafted on rootstocks of either P. persica, P. dulcis, P. armeniaca, P. davidiana or their hybrids, imported from Türkiye, taking into account the available scientific information, including the technical information provided by Türkiye. All pests associated with the commodity were evaluated against specific criteria for their relevance for this opinion. Four quarantine pests (peach rosette mosaic virus, tomato ringspot virus, Anoplophora chinensis, Scirtothrips dorsalis) and 14 non-regulated pests (Hoplolaimus galeatus, Lasiodiplodia pseudotheobromae, Neoscytalidium dimidiatum, Neoscytalidium novaehollandiae, Didesmococcus unifasciatus, Euzophera semifuneralis, Lepidosaphes malicola, Lepidosaphes pistaciae, Maconellicoccus hirsutus, Malacosoma parallela, Nipaecoccus viridis, Phenacoccus solenopsis, Pochazia shantungensis, Russellaspis pustulans) that fulfilled all relevant criteria were selected for further evaluation. For these 18 pests, the risk mitigation measures proposed in the technical Dossier from Türkiye were evaluated taking into account the possible limiting factors. For the selected pests, an expert judgement is given on the likelihood of pest freedom taking into consideration the risk mitigation measures acting on the pest, including uncertainties associated with the assessment. The degree of pest freedom varies among the pests evaluated, with fungi from Botryosphaeriaceae family (L. pseudotheobromae, N. dimidiatum and N. novaehollandiae) being the pests most frequently expected on the imported plants. The Expert Knowledge Elicitation indicated with 95% certainty that between 9,813 and 10,000 bundles (consisting of 10 or 25 plants each) per 10,000 would be free from the above-mentioned fungi in the Botryosphaeriaceae family.

Commodity risk assessment of Prunus domestica plants from Ukraine.

The European Commission requested the EFSA Panel on Plant Health to prepare and deliver risk assessments for commodities listed in Commission Implementing Regulation (EU) 2018/2019 as 'High risk plants, plant products and other objects'. This Scientific Opinion covers plant health risks posed by plants of Prunus domestica grafted on Prunus cerasifera imported from Ukraine, taking into account the available scientific information, including the technical information provided by Ukraine. All pests associated with the commodity were evaluated against specific criteria for their relevance for this opinion. One quarantine pest (Lopholeucaspis japonica), two protected zone quarantine pests (Erwinia amylovora and Xanthomonas arboricola pv. pruni) and one non-regulated pest (Eotetranychus prunicola) that fulfilled all relevant criteria were selected for further evaluation. For these four pests, the risk mitigation measures proposed in the technical dossier from Ukraine were evaluated taking into account the possible limiting factors. For the selected pests, an expert judgement is given on the likelihood of pest freedom taking into consideration the risk mitigation measures acting on the pest, including uncertainties associated with the assessment. The degree of pest freedom varies among the pests evaluated, with Xanthomonas arboricola pv. pruni being the pest most frequently expected on the imported plants. The Expert Knowledge Elicitation indicated with 95% certainty that between 9,870 and 10,000 bundles (consisting of 10 plants each) per 10,000 would be free from Xanthomonas arboricola pv. pruni.

Commodity risk assessment of Malus domestica plants from Turkey.

The European Commission requested the EFSA Panel on Plant Health to prepare and deliver risk assessments for commodities listed in Commission Implementing Regulation (EU) 2018/2019 as 'High risk plants, plant products and other objects'. This Scientific Opinion covers plant health risks posed by dormant grafted plants, rootstocks, budwood and scions of Malus domestica imported from Turkey, taking into account the available scientific information, including the technical information provided by Turkey. All pests associated with the commodities were evaluated against specific criteria for their relevance for this opinion. Three quarantine pests (Anoplophora chinensis, Lopholeucaspis japonica and tomato ringspot virus), one protected zone quarantine pest (Erwinia amylovora) and eight non-regulated pests (Calepitrimerus baileyi, Cenopalpus irani, Cicadatra persica, Diplodia bulgarica, Hoplolaimus galeatus, Malacosoma parallela, Pratylenchus loosi and Pyrolachnus pyri) that fulfilled all relevant criteria were selected for further evaluation. For E. amylovora, special requirements are specified in Commission Implementing Regulation (EU) 2019/2072. Based on the information provided in the dossier, the specific requirements for E. amylovora were not met. For Anoplophora chinensis, special measures are specified in Commission Implementing Decision (EU) 2012/138. The exporting country does meet the requirement for a certificate regarding plants for planting that originate from Turkish provinces other than Istanbul. For the 10 remaining selected pests, the risk mitigation measures proposed in the technical dossier from Turkey were evaluated taking into account the possible limiting factors. For the selected pests an expert judgement is given on the likelihood of pest freedom taking into consideration the risk mitigation measures acting on the pest, including uncertainties associated with the assessment. The degree of pest freedom varies among the pests evaluated, with D. bulgarica being the pest most frequently expected on the imported plants. The expert knowledge elicitation indicated with 95% certainty that between 9,863 and 10,000 bundles (consisting of 10 or 25 plants each) per 10,000 would be free from D. bulgarica.

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.

Going Viral: Virus-Based Biological Control Agents for Plant Protection.

The most economically important biotic stresses in crop production are caused by fungi, oomycetes, insects, viruses, and bacteria. Often chemical control is still the most commonly used method to manage them. However, the development of resistance in the different pathogens/pests, the putative damage on the natural ecosystem, the toxic residues in the field, and, thus, the contamination of the environment have stimulated the search for saferalternatives such as the use of biological control agents (BCAs). Among BCAs, viruses, a major driver for controlling host populations and evolution, are somewhat underused, mostly because of regulatory hurdles that make the cost of registration of such host-specific BCAs not affordable in comparison with the limited potential market. Here, we provide a comprehensive overview of the state of the art of virus-based BCAs against fungi, bacteria, viruses, and insects, with a specific focus on new approaches that rely on not only the direct biocidal virus component but also the complex ecological interactions between viruses and their hosts that do not necessarily result in direct damage to the host.

Commodity risk assessment of grafted plants of Malus domestica from Moldova.

The European Commission requested the EFSA Panel on Plant Health to prepare and deliver risk assessments for commodities listed in Commission Implementing Regulation (EU) 2018/2019 as 'High risk plants, plant products and other objects'. This Scientific Opinion covers plant health risks posed by defoliated and in dormant phase, grafted bare rooted plants for planting of Malus domestica imported from Moldova, taking into account the available scientific information, including the technical information provided by the applicant country. A list of 1,118 pests potentially associated with the commodity species was compiled. The relevance of these pests was assessed following defined criteria and based on evidence. The EU-quarantine pest Xiphinema rivesi non-EU populations fulfilled these criteria and was selected for further evaluation. For this pest, the risk mitigation measures proposed in the technical dossier from Moldova were evaluated taking into account the possible limiting factors. For this pest, an expert judgement is given on the likelihood of pest freedom taking into consideration the risk mitigation measures acting on it, including uncertainties associated with the assessment. The Expert Knowledge Elicitation indicated, with 95% certainty, that between 9,991 and 10,000 plants per 10,000 would be free of X. rivesi.

Leafhopper feeding behaviour on three grapevine cultivars with different susceptibilities to Flavescence dorée.

Scaphoideus titanus (Ball) is a grapevine-feeder leafhopper, and the most important vector of Flavescence dorée of grapevine (FD), a disease associated with phytoplasmas belonging to ribosomal subgroups 16Sr-V-C and -D. FD is a major constraint to viticulture in several European countries and, so far, its control has relied on roguing of infected plants and insecticide applications against the vector. Detailed knowledge on different levels of the multifaceted phytoplasma-plant-vector relationship is required to envisage and explore more sustainable ways to control the disease spread. In the present work, S. titanus feeding behaviour was described on three grapevine cultivars: Barbera (susceptible to FD), Brachetto, and Moscato (tolerant to FD) using the Electrical Penetration Graph (EPG) technique. Interestingly, no differences were highlighted in the non-phloem feeding phases, thus suggesting that the tested cultivars have no major differences in the biochemical composition or structure of the leaf cuticle, epidermis or mesophyll, that can affect the first feeding activities. On the contrary, the results showed significant differences in leafhopper feeding behaviour in terms of the duration of the phloem feeding phase, longer on Barbera and shorter on Brachetto and Moscato, and of the frequency of interruption-salivation events inside the phloem, higher on Brachetto and Moscato. These findings indicate a possible preference for the Barbera cultivar, a better host for the leafhopper. Scaphoideus titanus feeding behaviour on Barbera correlates with an enhanced FDp transmission efficiency, thus explaining, at least in part, the higher susceptibility of this cultivar to FD. The mechanisms for the possible non-preference for Brachetto and Moscato are discussed, and an antixenosis is hypothesized. We propose that breeding for resistance against FD should take into account both plant traits associated with the response to the phytoplasmas and to the vector.

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.

Commodity risk assessment of Malus domestica plants from Ukraine.

The European Commission requested the EFSA Panel on Plant Health to prepare and deliver risk assessments for commodities listed in Commission Implementing Regulation (EU) 2018/2019 as 'High risk plants, plant products and other objects'). This Scientific Opinion covers plant health risks posed by 1- to 3-year-old dormant grafted plants and rootstocks of Malus domestica imported from Ukraine, taking into account the available scientific information, including the technical information provided by Ukraine. All pests associated with the commodity were evaluated against specific criteria for their relevance for this opinion. Two quarantine pests (Lopholeucaspis japonica and Tobacco ringspot virus), one protected zone quarantine pest (Erwinia amylovora) and one non-regulated pest (Eotetranychus prunicola) that fulfilled all relevant criteria were selected for further evaluation. For Erwinia amylovora, for which special requirements are specified in Commission Implementing Regulation (EU) 2019/2072, Annex X, item 9, the fulfilment of these requirements was evaluated. Based on the information provided in the dossier, the specific requirements for Erwinia amylovora were not met. For the three remaining selected pests, the risk mitigation measures proposed in the technical dossier from Ukraine were evaluated taking into account the possible limiting factors. For the selected pests, an expert judgement is given on the likelihood of pest freedom taking into consideration the risk mitigation measures acting on the pest, including uncertainties associated with the assessment. The degree of pest freedom varies among the pests evaluated, with Eotetranychus prunicola being the pest most frequently expected on the imported plants. The Expert Knowledge Elicitation indicated with 95% certainty that between 9,912 and 10,000 bundles (consisting of 50 plants each) per 10,000 would be free from Eotetranychus prunicola.

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.

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.

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.

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.