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Abstract 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 avium possibly grafted on rootstocks of either P. avium, P. canescens, P. cerasus, P. pseudocerasus or their hybrids 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. Three quarantine pests Scirtothrips dorsalis, tobacco ringspot virus and tomato ringspot virus), one protected zone EU quarantine pest (Bemisia tabaci (European population), and three non‐ regulated pests (Colletotrichum aenigma, Eulecanium excrescens and Takahashia japonica) that fulfilled all relevant criteria were selected for further evaluation. 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 Colletotrichum aenigma being the pest most frequently expected on the imported potted plants. The Expert Knowledge Elicitation indicated with 95% certainty that between 9971 and 10,000 plants per 10,000 would be free from the above‐mentioned fungus.

Abstract 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.

Abstract 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.

Abstract 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.

Abstract 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.

Abstract 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.

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.

Abstract 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.

Abstract 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 12 non‐regulated pests (Calepitrimerus baileyi, Cenopalpus irani, Cicadatra persica, Didesmococcus unifascuiatus, Diplodia bulgarica, Euzophera semifuneralis, Hoplolaimus galeatus, Maconellicoccus hirsutus, Malacosoma parallela, Pochazia shantungensis, Pratylenchus loosi, Russellaspis pustulans 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 14 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.

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.

Abstract 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.

Abstract 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.

Abstract 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.

Abstract 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.

Abstract 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.

Abstract 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.

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 β 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 β 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.

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.

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.

Flavescence dorée (FD) is a quarantine grapevine disease caused by a phytoplasma transmitted by the leafhopper Scaphoideus titanus Ball. FD management relies on compulsory insecticide treatments, roguing of infected plants, and substitution with certified material. Some grapevine cultivars show a spontaneous remission of symptoms (recovery). To determine if recovery is a suitable strategy to co-exist with disease in areas of strong infestation, the qualitative aspects of grapes, musts, and wines obtained from recovered Barbera and Chardonnay grapevines were investigated in two productive vineyards. Following field observations, about 1500 plants in each vineyard were divided into healthy (asymptomatic and negative in phytoplasma molecular diagnosis) and recovered (asymptomatic the year of observation but infected the year before). Maturation curves and microvinification tests followed by oenological and sensory analyses showed that maturation trends of recovered grapes were in line with those from healthy plants and the final qualities of wines were comparable. The spread of FD has strongly increased in Piedmont (Italy) in recent decades. Management strategies to cope with the disease are necessary to preserve traditional wine production. Despite the yield from recovered grapevines is quantitatively lower than that from healthy ones, we showed here that the wine quality is, however, preserved.

Quantitative estimates of vector populations and their infectivity in the wild and in cultivated compartments of agroecosystems have been carried out to elucidate the role of the wild compartment in the epidemiology of Flavescence dorée (FD). Seven sites were selected for the investigations in the Piedmont Region of Italy. They were characterized by a high variety of agricultural and ecological landscape features, and included a vineyard surrounded by wild vegetation. In order to describe abundance and prevalence of FD-infected vectors in the cultivated and wild compartments of the vineyard agroecosystem, adults of Scaphoideus titanus were collected by yellow sticky traps inside and outside the vineyard over the period July 10th–September 9th, 2015. They were counted and singly analyzed for the presence of FD phytoplasmas by PCR. Multifactorial correlations among vector population level, prevalence of infected insects inside and outside the vineyards, disease prevalence in cultivated and wild Vitis plants, and location of wild Vitis plants with respect to the vineyard were analyzed. Abundance of S. titanus adults significantly decreased from the end of July onwards, particularly inside the vineyard (average range 22.7 ± 2.5 insects/trap). Percentage of FD-positive S. titanus was significantly higher outside the vineyard (up to 48% on average) compared to inside the vineyard (up to 34% on average), and increased during the season in both compartments.

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.

Micro(mi)RNAs play crucial roles in plant developmental processes and in defense responses to biotic and abiotic stresses. In the last years, many works on small RNAs in grapevine (Vitis spp.) were published, and several conserved and putative novel grapevine-specific miRNAs were identified. In order to reorganize the high quantity of available data, we produced “miRVIT,” the first database of all novel grapevine miRNA candidates characterized so far, and still not deposited in miRBase. To this aim, each miRNA accession was renamed, repositioned in the last version of the grapevine genome, and compared with all the novel and conserved miRNAs detected in grapevine. Conserved and novel miRNAs cataloged in miRVIT were then used for analyzing Vitis vinifera plants infected by Flavescence dorée (FD), one of the most severe phytoplasma diseases affecting grapevine. The analysis of small RNAs from healthy, recovered (plants showing spontaneous and stable remission of symptoms), and FD-infected “Barbera” grapevines showed that FD altered the expression profiles of several miRNAs, including those involved in cell development and photosynthesis, jasmonate signaling, and disease resistance response. The application of miRVIT in a biological context confirmed the effectiveness of the followed approach, especially for the identification of novel miRNA candidates in grapevine. miRVIT database is available at http://mirvit.ipsp.cnr.it.Highlights: The application of the newly produced database of grapevine novel miRNAs to the analysis of plants infected by Flavescence dorée reveals key roles of miRNAs in photosynthesis and jasmonate signaling.

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.

Phytoplasmas are plant pathogenic bacteria, naturally transmitted by insects and confined in the phloem of the host plant, where they take up nutrients and eventually cause plant death. Their control is mainly based on insecticide treatments. The aim of this work was to study the effect of two AM fungi in modifying plant response to chrysanthemum yellows phytoplasma (CY) infection in chrysanthemum plants. Inoculation of Glomus intraradices BB-E and G. mosseae BEG12 reduced the damage caused by this plant pathogen in the aerial part of the plant, increased plant tolerance to the infection and reduced the severity of symptom expression, probably in a systemic way. Inoculation with G. mosseae did not alter CY multiplication and viability in young leaves, whilst the morphology of CY mature leaves was typical of senescent cells. Possible mechanisms involved are discussed.

Phytoplasmas, uncultivable phloem-limited phytopathogenic wall-less bacteria, represent a major threat to agriculture worldwide. They are transmitted in a persistent, propagative manner by phloem-sucking Hemipteran insects. Phytoplasma membrane proteins are in direct contact with hosts and are presumably involved in determining vector specificity. Such a role has been proposed for phytoplasma transmembrane proteins encoded by circular extrachromosomal elements, at least one of which is a plasmid. Little is known about the interactions between major phytoplasma antigenic membrane protein (Amp) and insect vector proteins. The aims of our work were to identify vector proteins interacting with Amp and to investigate their role in transmission specificity. In controlled transmission experiments, four Hemipteran species were identified as vectors of "Candidatus Phytoplasma asteris", the chrysanthemum yellows phytoplasmas (CYP) strain, and three others as non-vectors. Interactions between a labelled (recombinant) CYP Amp and insect proteins were analysed by far Western blots and affinity chromatography. Amp interacted specifically with a few proteins from vector species only. Among Amp-binding vector proteins, actin and both the α and β subunits of ATP synthase were identified by mass spectrometry and Western blots. Immunofluorescence confocal microscopy and Western blots of plasma membrane and mitochondrial fractions confirmed the localisation of ATP synthase, generally known as a mitochondrial protein, in plasma membranes of midgut and salivary gland cells in the vector Euscelidius variegatus. The vector-specific interaction between phytoplasma Amp and insect ATP synthase is demonstrated for the first time, and this work also supports the hypothesis that host actin is involved in the internalization and intracellular motility of phytoplasmas within their vectors. Phytoplasma Amp is hypothesized to play a crucial role in insect transmission specificity.

Tomato plants (Lycopersicon esculentum Mill.) showing leaf yellowing or reddening, proliferation of lateral shoots, hypertrophied calyces, and greening of petals were found in four tomato-growing areas of central Italy during surveys carried out in 1999–2000. The highest disease incidence was recorded in 1999 (78%) and 2000 (82%) near Ronciglione (Viterbo province). The phytoplasmas associated with the disease were detected and characterised using PCR and RFLP. They belonged to 16S rRNA groups I, III, V and XII (sensu Lee et al., 1998. Int. J. Syst. Bacteriol., 48, 1153). Forty-five percent of tomato plants were infected with group I (max. 52% near Frascati and Ronciglione), 31% with group XII (max. 39% near Ronciglione) and 24% with other groups (max. 33% near Tarquinia and 23% near Fondi). Seven percent of tomato plants were infected with both group I and group XII. This is the first report of a group-III and a group-V phytoplasma found in tomato.