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Phytoplasmas are plant-pathogenic Mollicutes transmitted by leafhoppers, planthoppers, and psyllids in a persistent propagative manner. Chrysanthemum yellows phytoplasma (CY) is a member of ‘ Candidatus Phytoplasma asteris’, 16Sr-IB, and is transmitted by at least three leafhopper species, Macrosteles quadripunctulatus Kirschbaum, Euscelidius variegatus Kirschbaum, and Euscelis incisus Kirschbaum (all Homoptera: Cicadellidae: Deltocephalinae). Although M. quadripunctulatus transmits CY with very high efficiency (near 100%), 25% of E. variegatus repeatedly fail to transmit CY. The aims of this work were to correlate vector ability with different pathogen distribution in the insect body and to investigate the role of midgut and salivary glands as barriers to CY transmission. Euscelidius variegatus individuals acquired CY by feeding on infected plants or by abdominal microinjection of a phytoplasma-enriched suspension. Insects were individually tested for transmission on daisy seedlings [ Chrysanthemum carinatum Schousboe (Asteraceae)], and thereafter analysed by real-time polymerase chain reaction (PCR) for CY concentration on whole insects or separately on heads and the rest of the body. Hoppers were classified as early and late transmitters or non-transmitters, according to the time inoculated plants required for expression of CY symptoms. Similar transmission efficiencies were achieved following feeding or abdominal microinjection, suggesting that salivary glands may be a major barrier to transmission. Following acquisition from infected plants, all transmitters tested positive by PCR, and 60% of non-transmitters also tested positive although with a significantly lower CY concentration. This indicates that a minimum number of phytoplasma cells may be required for successful transmission. The midgut may have prevented phytoplasma entry into the haemocoel of PCR-negative non-transmitters. Results suggest that both midgut and salivary glands may act as barriers. To assess the effect on CY transmission of a specific parasitic bacterium of E. variegatus, tentatively named BEV (Bacterium Euscelidius variegatus), we established a BEV-infected population by abdominal microinjection of BEV bacteria. The presence of BEV did not significantly alter the efficiency of CY transmission. [ABSTRACT FROM AUTHOR]

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., (© 2020 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

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., (© 2020 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

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., (© 2020 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

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., (© 2020 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

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.

The study of phytoplasma membrane protein interactions with host cell proteins is crucial to understand the life cycle of these unculturable microorganisms within their hosts. A step-by-step protocol for the heterologous expression of phytoplasma membrane proteins in Escherichia coli is described, together with a procedure to purify a suitable quantity of fusion antigen for application in the study of phytoplasma-host interactions.