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During field surveys conducted in 2020 in Jordanian orchards, phytoplasma‐like symptoms (leaf yellowing/reddening and rolling, and witches'‐broom) were observed in three stone fruit species (peach, European plum, sweet cherry) and persimmon. Molecular analyses identified phytoplasma strains belonging to the species 'Candidatus Phytoplasma solani' (subgroup 16SrXII‐A) as largely prevalent in stone fruit and persimmon symptomatic plants. Moreover, 'Ca. Phytoplasma omanense' (16SrXXIX‐B) was found in few European plum symptomatic plants. In previous studies, such phytoplasma strains were identified in other important crops (almond, pomegranate, and grapevine) and in several putative insect vectors, suggesting their complex ecology in Jordan. Further studies are needed to in‐depth investigate the diffusion of phytoplasma‐associated diseases of stone fruits throughout the Country, to clarify their aetiology, and to study their epidemiological pattern(s). [ABSTRACT FROM AUTHOR]

Field surveys were conducted in wine and table grape vineyards from June to October 2020 in 13 locations belonging to five governorates in North and South Jordan. Typical grapevine yellows symptoms, including leaf reddening/yellowing and rolling were observed on 10% to 55% of vines. Nested PCR‐based amplification of the 16S rRNA gene detected phytoplasmas in 22% and 15.7% of the analysed symptomatic wine and table grape cultivar plants, respectively. Amplicon nucleotide sequence analyses identified the detected phytoplasmas as "Candidatus Phytoplasma solani" (taxonomic subgroup 16SrXII‐A), "Ca. P. omanense" (16SrXXIX‐A and ‐B), "Ca. P. aurantifolia" (16SrII‐C) and "Ca. P. asteris" (16SrI‐R) in 72.4%, 17.2%, 6.9% and 3.4% of infected plants, respectively. Such phytoplasmas were found differentially distributed in wine and table grape cultivar vineyards surveyed. Further investigation identified "Ca. P. solani" in the putative insect vectors Orosius cellulosus (first report in Jordan), Euscelidius mundus, Laodelphax striatellus, and Circulifer sp., and in bindweed; "Ca. P. aurantifolia" in the insect O. cellulosus and in bindweed; "Ca. P. omanense" in the insect Psammotettix striatus; and "Ca. P. asteris" in the insects Arboridia adanae, Cicadulina bipunctata, Circulifer sp., L. striatellus, Hyalesthes obsoletus, and P. striatus. Based on this preliminary data, ecological cycles of such phytoplasmas are discussed. Results suggest that the diversity and ecology of grapevine yellows phytoplasmas in Jordan are more complex than previously known, leading to a potential risk of disease outbreaks. [ABSTRACT FROM AUTHOR]

During field surveys conducted in northern Jordan from June to November 2020, phytoplasma‐like symptoms, including leaf yellowing/reddening and rolling, little leaf and witches' broom were observed in pomegranate. Disease incidence in 22 surveyed orchards ranged from 30% to 65%. Nested PCR‐based amplification of 16S rRNA gene detected phytoplasmas in 17% of collected symptomatic pomegranate trees. Amplicon nucleotide sequence analyses allowed attributing the detected phytoplasmas to 'Candidatus Phytoplasma solani', 'Ca. P. aurantifolia', 'Ca. P. asteris' and 'Ca. P. ulmi'. These phytoplasmas were found in plants showing specific symptoms and differentially distributed in the considered locations. Additionally, three cicadellids (Macrosteles sexnotatus, Cicadulina bipunctata and Psammotettix striatus) and two non‐crop plants (Plantago major and Capsicum annuum) resulted hosting 'Ca. P. asteris' strains, and one cicadellid (Balclutha incisa) was carrying a 'Ca. P. solani' strain. A new pomegranate disease complex associated with multiple phytoplasmas, including 'Ca. P. aurantifolia' and 'Ca. P. ulmi', never reported before in this host plant, is described here. Moreover, preliminary indications are provided on its possible epidemiology in Jordan, involving two putative insect vectors (M. sexnotatus, B. incisa) first reported in the Country. [ABSTRACT FROM AUTHOR]

Orientus ishidae (Matsumura) (subfamily Deltocephalinae) is an Asian species now widespread in Europe, and a vector of 16SrV phytoplasmas agents of grapevine Flavescence dorée (FDP). Embryonic and post-embryonic development, spatial distribution, and relationships with grapevine of nymphs were studied under field and laboratory conditions. Egg-hatching dynamics and post-embryonic development of nymphs were studied by collecting grapevine wood from managed and unmanaged vineyards (including bot European Vitis vinifera L. and wild American rootstocks) and storing it inside rearing cages at T = 21–23°C. Field sampling of nymphs were made on both grapevine and two elective host plants of O. ishidae: hazelnut and hornbeam. Taylor's Power Law was applied to assess the aggregation coefficient of early- (first and second) and late- (third to fifth) life instars on leaves and shoots of host plants. More nymphs were obtained from wood collected in unmanaged rather than managed vineyards. Under lab conditions, the embryonic development lasted 34–48 d, whereas the whole post-embryonic development averaged 27 d. Under field conditions, early instars peaked at the end of May, and late instars peaked 2–4 wk later. The aggregation patterns decreased from early to late instars, and from leaves to shoots. Very few nymphs were observed on unmanaged grapevine, either European or American, and none on managed European grapevine. Some behavioral and FDP epidemiological consequences of the results obtained are discussed. [ABSTRACT FROM AUTHOR]

Two of the most common leafhoppers present in Chile are the Cicadelli - dae Paratanus exitiosus and Bergallia valdiviana . They commonly occur in vineyards of central Chile, including some vineyards infected by phytoplasmas. The present study demonstrates that P. exitiosus and B. valdiviana can transmit 16SrIII-J phytoplasmas to grapevine and periwinkle plants. This provides improved understanding of the 16SrIII- J phytoplasma epidemiology in Chilean vineyards. [ABSTRACT FROM AUTHOR]

Insect immunity is a crucial process in interactions between host and microorganisms and the presence of pathogenic, commensal, or beneficial bacteria may result in different immune responses. In Hemiptera vectors of phytoplasmas, infected insects are amenable to carrying high loads of phytopathogens, besides hosting other bacterial affiliates, which have evolved different strategies to be retained; adaptation to host response and immunomodulation are key aspects of insect-symbiont interactions. Most of the analyses published to date has investigated insect immune response to pathogens, whereas few studies have focused on the role of host immunity in microbiota homeostasis and vectorial capacity. Here the expression of immune genes in the leafhopper vector of phytoplasmas Euscelidius variegatus was investigated following exposure to Asaia symbiotic bacteria, previously demonstrated to affect phytoplasma acquisition by leafhoppers. The expression of four genes related to major components of immunity was measured, i.e., defensin, phenoloxidase, kazal type 1 serine protease inhibitor and Raf, a component of the Ras/Raf pathway. The response was separately tested in whole insects, midguts and cultured hemocytes. Healthy individuals were assessed along with specimens undergoing early- and late-stage phytoplasma infection. In addition, the adhesion grade of Asaia strains was examined to assess whether symbionts could establish a physical barrier against phytoplasma colonization. Our results revealed a specific activation of Raf in midguts after double infection by Asaia and flavescence dorée phytoplasma. Increased expression was observed already in early stages of phytoplasma colonization. Gut-specific localization and timing of Raf activation are consistent with the role played by Asaia in limiting phytoplasma acquisition by E. variegatus , supporting the involvement of this gene in the anti-pathogen activity. However, limited attachment capability was found for Asaia under in vitro experimental conditions, suggesting a minor contribution of physical phytoplasma exclusion from the vector gut wall. By providing evidence of immune modulation played by Asaia , these results contribute to elucidating the molecular mechanisms regulating interference with phytoplasma infection in E. variegatus. The involvement of Raf suggests that in the presence of reduced immunity (reported in Hemipterans), immune genes can be differently regulated and recruited to play additional functions, generally played by genes lost by hemipterans. [ABSTRACT FROM AUTHOR]

Some pathogenic phloem‐limited bacteria are a major threat for worldwide agriculture due to the heavy economic losses caused to many high‐value crops. These disease agents – phytoplasmas, spiroplasmas, liberibacters, and Arsenophonus‐like bacteria – are transmitted from plant to plant by phloem‐feeding Hemiptera vectors. The associations established among pathogens and vectors result in a complex network of interactions involving also the whole microbial community harboured by the insect host. Interactions among bacteria may be beneficial, competitive, or detrimental for the involved microorganisms, and can dramatically affect the insect vector competence and consequently the spread of diseases. Interference is observed among pathogen strains competing to invade the same vector specimen, causing selective acquisition or transmission. Insect bacterial endosymbionts are another pivotal element of interactions between vectors and phytopathogens, because of their central role in insect life cycles. Some symbionts, either obligate or facultative, were shown to have antagonistic effects on the colonization by plant pathogens, by producing antimicrobial substances, by stimulating the production of antimicrobial substances by insects, or by competing for host infection. In other cases, the mutual exclusion between symbiont and pathogen suggests a possible detrimental influence on phytopathogens displayed by symbiotic bacteria; conversely, examples of microbes enhancing pathogen load are available as well. Whether and how bacterial exchanges occurring in vectors affect the relationship between insects, plants, and phytopathogens is still unresolved, leaving room for many open questions concerning the significance of particular traits of these multitrophic interactions. Such complex interplays may have a serious impact on pathogen spread and control, potentially driving new strategies for the containment of important diseases. Phloem‐limited bacterial phytopathogens, transmitted among plants by phloem‐feeding Hemiptera vectors, cause heavy losses in agriculture. Associations of pathogens and vectors form complex networks of interactions involving also the microbial community harboured by the insect host; bacterial interactions may have beneficial or detrimental influences on a vector's efficacy. How such complex interplays affect insect‐plant‐bacteria interactions is still unresolved; however, their impact on pathogen spread and control could drive new strategies for the containment of important diseases. [ABSTRACT FROM AUTHOR]

The transmission of microbial pathogens by insect vectors can be affected by the insect’s microbial symbionts, which may compete in colonizing organs, express antagonistic factors or activate host immune response. Acetic acid bacteria of the genus Asaia are symbionts of the leafhopper Scaphoideus titanus, which transmits Flavescence dorée phytoplasma. These bacteria could be used as control agents against the disease. Here, we experimentally investigated the interaction between different strains of Asaia and phytoplasma transmission in the laboratory by using the model leafhopper Euscelidius variegatus and the plant host Vicia faba. We found that uncultivable and low concentrations of Asaia phylotypes were associated with E. variegatus. When we supplied different Asaia strains isolated from other insects and exhibiting different phenotypes to E. variegatus orally, the bacteria stably colonized the leafhopper, reached relatively higher densities and could then be isolated from the host. We conducted transmission trials of Flavescence dorée phytoplasma with individuals colonized with three exogenous Asaia strains. When the phytoplasma became established in the bodies of E. variegatus, leafhoppers were able to transmit it to broad beans, with transmission rates ranging from 33 to 76% in different experiments. However, leafhoppers that were colonized by one of the Asaia strains producing an air-liquid interface biofilm exhibited significantly reduced phytoplasma acquisition, with infection rates at 5-28%, whereas they were 25-77% in control insects. Although the mechanisms regulating this interference remain to be elucidated, our results provide evidence of the potential use of Asaia as a biocontrol agent. [ABSTRACT FROM AUTHOR]

The possible colonization of vineyards by leafhoppers, planthoppers and their allies, known or suspected vectors of phytoplasmas to vines, living on adjacent foraging strip crops seeded for pollinators was evaluated in Piedmont, NW Italy. Strip crops consisted in a seed mixture of Fabaceae and in rape (Brassica napus var. oleifera L.). Insects were collected with a sweep net on the strips, in the vineyard inter-row (close to and far from the strip), and in the surrounding habitat. DNA was extracted from single specimens, and PCR was performed to identify phytoplasmas of different groups, with a particular emphasis on groups 16SrV and 16SrXII. A one-way ANOVA was made for detecting differences in catches between strips and vineyards'inter-rows of the most abundant species. Among vectors, Neoaliturus fenestratus (Herrich-Schaffer), Euscelis incisus (Kirschbaum) and Dictyophara europaea (L.) were locally abundant as a consequence of the presence of weeds serving as host plants. Phytoplasmas of groups 16SrV and 16SrIX were identified in three specimens of D. europaea and one of N. fenestratus, respectively. D. europaea was more abundant on the strips, whereas the patterns of N. fenestratus changed from site to site. Concerning other species (not associated to 16SrV and 16SrXII phytoplasmas), high levels of Psammotettix alienus (Dalbholm) and Philaenus spumarius (L.) were observed. The presence of a flowering strip close to a vineyard does not seem to be a threat, if a good ground cover is achieved by the seed mixture. Otherwise, the development of certain weeds may cause an increase of leafhopper species known or suspected vectors of phytoplasmas. [ABSTRACT FROM AUTHOR]

During field surveys carried out from June to October 2020 and in January 2021 in orchards of northern Jordan, phytoplasma-like symptoms (early flowering along with evergreen pattern; witches'-broom, yellowing, and dieback; slim leaf and leaf rolling; stem fasciation) were observed in almond trees. In 23 investigated orchards, symptomatic almond trees ranged from 20 to 85%. PCR-based amplification of 16S rRNA gene detected phytoplasmas in 21% of 140 collected symptomatic almond trees. Sequence analyses allowed attributing the detected phytoplasmas to ' Candidatus Phytoplasma asteris' (taxonomic subgroups 16SrI-B and -R), ' Ca. P. aurantifolia' (16SrII-B and -C), ' Ca. P. omanense' (16SrXXIX-A and -B), ' Ca. P. phoenicium' (16SrIX-B), ' Ca. P. pyri' (16SrX-C), ' Ca. P. solani' (16SrXII-A), and ' Ca. P. ulmi' (16SrV-A). Such phytoplasmas were found associated with specific symptoms and differentially distributed in the considered locations. Moreover, further investigation identified ' Ca. P. asteris' (subgroup 16SrI-R) in putative insect vectors such as Empoasca sp., Reptalus sp., and Hyalesthes obsoletus , ' Ca. P. pyri' in Cacopsylla bidens , and ' Ca. P. omanense' (subgroup 16SrXXIX-B) in the non-crop plant Amaranthus sp. In conclusion, this study described an almond disease complex associated with multiple phytoplasmas, including ' Ca. P. pyri', ' Ca. P. omanense', and ' Ca. P. ulmi' that were never reported before in this crop. Further studies are needed to survey the diffusion of this almond disease complex in the region, demonstrate the transmission capability of the identified putative vectors, and in-depth investigate the ecologies of all phytoplasmas associated with the disease. • Seven ʻ Candidatus Phytoplasmaʼ species were reported in Jordan in association with an almond disease complex. • ʻ Ca. P. omanenseʼ, ʻ Ca. P. ulmi', and ʻ Ca. P. pyriʼ were firstly reported in almond around the world. • New subgroup XXIX-B was described. • Eight insect taxa were found positive to ʻ Ca. Phytoplasma pyriʼ and ʻ Ca. P. asterisʼ. [ABSTRACT FROM AUTHOR]

Psyllids, as vectors of phloem-restricted plant pathogens, are serious agricultural pests. Fruit tree phytoplasmas are transmitted by different Cacopsylla spp., while other psyllids are known vectors of liberibacters. Recently, the bacterium ' Candidatus Liberibacter europaeus' was found in pear trees and in Cacopsylla pyri (Linnaeus), the vector of ' Ca. Phytoplasma pyri'. This new species does not cause symptoms in plants and is probably a symbiont rather than a pathogen. Based on these findings and the assumption that ' Ca. Liberibacter europaeus' is widespread, we studied its distribution in the genus Cacopsylla and in the respective host and shelter plants (where psyllids aestivate and overwinter), as well as its possible co-presence with ' Ca. Phytoplasma' spp. We tested 14 Cacopsylla species and 11 plant species from northwestern Italy, Hungary and Israel, characterized by warm oceanic, temperate continental and warm Mediterranean climatic conditions, respectively. ' Ca. Liberibacter europaeus' was common within the Cacopsylla genus, being present in nine of the 14 species screened as well as in most host plants, whereas none of the shelter plants tested positive for this bacterium. Altogether, these findings indicate the presence of ' Ca. Liberibacter europaeus' in continental zones, whereas it does not seem to be widespread in the Mediterranean region. Furthermore, lack of specific symptoms in all infected plants confirms an endophytic relationship with this bacterium, while its abundance in insects suggests a beneficial role for the host. Co-infections with phytoplasmas, observed in insects and plants, require further study to evaluate the possible interactions between them. [ABSTRACT FROM AUTHOR]

Background: Bacteria of the genus Asaia have been recently recognized as secondary symbionts of different sugar-feeding insects, including the leafhopper Scaphoideus titanus, vector of Flavescence dorée phytoplasmas. Asaia has been shown to be localized in S. titanus gut, salivary glands and gonoducts and to be maternally transmitted to the progeny by an egg smearing mechanism. It is currently not known whether Asaia in S. titanus is transmitted by additional routes. We performed a study to evaluate if Asaia infection is capable of horizontal transmission via co-feeding and venereal routes. Results: A Gfp-tagged strain of Asaia was provided to S. titanus individuals to trace the transmission pathways of the symbiotic bacterium. Co-feeding trials showed a regular transfer of bacterial cells from donors to recipients, with a peak of frequency after 72 hours of exposure, and with concentrations of the administrated strain growing over time. Venereal transmission experiments were first carried out using infected males paired with uninfected females. In this case, female individuals acquired Gfp-labelled Asaia, with highest infection rates 72-96 hours after mating and with increasing abundance of the tagged symbiont over time. When crosses between infected females and uninfected males were conducted, the occurrence of "female to male" transmission was observed, even though the transfer occurred unevenly. Conclusions: The data presented demonstrate that the acetic acid bacterial symbiont Asaia is horizontally transmitted among S. titanus individuals both by co-feeding and venereal transmission, providing one of the few direct demonstrations of such a symbiotic transfer in Hemiptera. This study contributes to the understanding of the bacterial ecology in the insect host, and indicates that Asaia evolved multiple pathways for the colonization of S. titanus body. [ABSTRACT FROM AUTHOR]

Apple proliferation (AP) is an important disease and is prevalent in several European countries. The causal agent of AP is ‘ Candidatus Phytoplasma mali’ (‘ Ca. Phytoplasma mali’). In this work, isolates of ‘ Ca. Phytoplasma mali’ were detected and characterized through polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analyses of 16S rRNA gene and non-ribosomal DNA fragment. The presence of three AP subtypes (AT-1, AT-2 and AP-15) was identified in 31 symptomatic apple trees and two samples each constituted by a pool of five insects, collected in north-western Italy, where AT-1 is a dominant subtype. Subsequent nucleotide sequence analysis of the PCR-amplified 1.8 kb (P1/P7) fragment, containing the 16S rDNA, the 16S–23S intergenic ribosomal region and the 5′-end of the 23S rDNA, revealed the presence of at least two phytoplasmal genetic lineages within the AT-1 subtype, designed AT-1a and AT-1b. Moreover, in silico single nucleotide polymorphism (SNP) analysis based on 16S rDNA sequence can differentiate AT-1 subtype from AT-2 and AP-15 subtypes. Our data showed a high degree of genetic diversity among ‘ Ca. Phytoplasma mali’ population in north-western Italy and underlined the possible use of the 16S rDNA analysis for the identification and the geographical origin assignation of isolates of AP phytoplasma. Molecular markers on 16S rDNA, here identified, could be useful for studying the epidemiology of AP disease. [ABSTRACT FROM AUTHOR]

Laboratory trials were carried out to transmit ‘Candidatus Phytoplasma mall’ to healthy apple seedlings with the leafhopper Fieberiella florii. Experiments on serial inoculation access period and molecular analyses performed on test plants and insects confirmed the ability of the leaf-hopper to carry and transmit the phytoplasma. Field surveys by means of yellow sticky traps were conducted in northwestern Italy to verify the abundance and the natural infectivity of F. florii in apple orchards and in wild vegetation in areas surrounding apple orchards. Despite the high percentages of infected specimens obtained in the apple orchards (5.7%) and in the wild vegetation areas (20.0%), the risk of apple tree infection by F. florii in nature is probably low because of the very low insect density recorded. In spite of the low number of specimens collected, the presence of the leafhopper in apple orchards in summer, when the main vector, the psyllid Cacopsylla melanoneura, feeds on alternative hosts, is meaningful. Moreover, the high degree of polyphagy of the leafhopper opens up new interesting prospects for the epidemiology of apple proliferation. [ABSTRACT FROM AUTHOR]

To date, phytoplasmas belonging to six ribosomal subgroups have been detected to infect grapevines in Chile in 36 percent of the sampled plants. A new survey on the presence of grapevine yellows was carried out from 2016 to 2020, and 330 grapevine plants from the most important wine regions of the country were sampled and analyzed by nested PCR/RFLP analyses. Phytoplasmas enclosed in subgroups 16SrIII-J and 16SrVII-A were identified with infection rates of 17% and 2%, respectively. The vineyards in which the phytoplasma-infected plants were detected were further inspected to identify alternative host plants and insects of potential epidemiological relevance. Five previously unreported plant species resulted positive for 16SrIII-J phytoplasma (Rosa spp., Brassica rapa, Erodium spp., Malva spp. and Rubus ulmifolius) and five insect species were fully or partially identified (Amplicephalus ornatus, A. pallidus, A. curtulus, Bergallia sp., Exitianus obscurinervis) as potential vectors of 16SrIII-J phytoplasmas. The 16SrVII-A phytoplasmas were not detected in non-grape plant species nor in insects. This work establishes updated guidelines for the study, management, and prevention of grapevine yellows in Chile, and in other grapevine growing regions of South America. [ABSTRACT FROM AUTHOR]

Intracellular reproductive manipulators, such as Candidatus Cardinium and Wolbachia are vertically transmitted to progeny but rarely show co-speciation with the host. In sap-feeding insects, plant tissues have been proposed as alternative horizontal routes of interspecific transmission, but experimental evidence is limited. Here we report results from experiments that show that Cardinium is horizontally transmitted between different phloem sap-feeding insect species through plants. Quantitative PCR and in situ hybridization experiments indicated that the leafhopper Scaphoideus titanus releases Cardinium from its salivary glands during feeding on both artificial media and grapevine leaves. Successional time-course feeding experiments with S. titanus initially fed sugar solutions or small areas of grapevine leaves followed by feeding by the phytoplasma vector Macrosteles quadripunctulatus or the grapevine feeder Empoasca vitis revealed that the symbionts were transmitted to both species. Explaining interspecific horizontal transmission through plants improves our understanding of how symbionts spread, their lifestyle and the symbiont-host intermixed evolutionary pattern. [ABSTRACT FROM AUTHOR]