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Despite several decades’ effort to detect and identify phytoplasmas (Mollicutes) using PCR and Sanger sequencing focusing on diseased plants, knowledge of phytoplasma biodiversity and vector associations remains highly incomplete. To improve protocols for documenting phytoplasma diversity and ecology, we used DNA extracted from phloem-feeding insects and compared traditional Sanger sequencing with a next-generation sequencing method, Anchored Hybrid Enrichment (AHE) for detecting and characterizing phytoplasmas. Among 22 of 180 leafhopper samples that initially tested positive for phytoplasmas using qPCR, AHE yielded phytoplasma 16Sr sequences for 20 (19 complete and 1 partial sequence) while Sanger sequencing yielded sequences for 16 (11 complete and 5 partial). AHE yielded phytoplasma sequences for an additional 7 samples (3 complete and 4 partial) that did not meet the qPCR threshold for phytoplasma positivity or yielded non-phytoplasma sequences using Sanger sequencing. This suggests that AHE is more efficient for obtaining phytoplasma sequences. Twenty-three samples with sufficient data were classified into eight 16Sr subgroups (16SrI-B, I-F, I-AO, III-U, V-C, IX-J, XI-C, XXXVII-A), three new subgroups (designated as 16SrVI-L, XV-D, XI-G) and three possible new groups. Our results suggest that screening phloem-feeding insects using qPCR and AHE sequencing may be the most efficient method for discovering new phytoplasmas. 

The Nearctic leafhopper species Gyponana (Gyponana) mali DeLong, 1942 isreported from Europe for the fi rst time and represents the fi rst record of the tribe Gyponini Stål, 1870 (Hemiptera: Cicadellidae: Iassinae: Gyponini) for the Palearctic Region. Specimens were collected in southern Switzerland (Ticino) and two regions of northern Italy (Lombardy and Veneto) in 2015–2019. The preferred host plant in these areas appears to be Cornus sanguinea L. Phylogenetic analysis of the COI barcode sequences grouped one of the European specimens with three individuals of G. (G.) mali from Ontario, Canada. Morphological study indicated that the male genitalia of the European population are intermediate between G. (G.) mali and G. (G.) extenda DeLong, 1942. 

Phytoplasmas are obligate transkingdom bacterial parasites that infect a variety of plant species and replicate in phloem-feeding insects in the order Hemiptera, mainly leafhoppers (Cicadellidae). The insect capacity in acquisition, transmission, survival, and host range directly determines the epidemiology of phytoplasmas. However, due to the difficulty of insect sampling and the lack of follow-up transmission trials, the confirmed phytoplasma insect hosts are still limited compared with the identified plant hosts. Recently, quantitative polymerase chain reaction (qPCR)-based quick screening of 227 leafhoppers collected in natural habitats unveiled the presence of previously unknown phytoplasmas in six samples. In the present study, 76 leafhoppers, including the six prescreened positive samples, were further examined to identify and characterize the phytoplasma strains by semi-nested PCR. A total of ten phytoplasma strains were identified in leafhoppers from four countries including South Africa, Kyrgyzstan, Australia, and China. Based on virtual restriction fragment length polymorphism (RFLP) analysis, these ten phytoplasma strains were classified into four distinct ribosomal (16Sr) groups (16SrI, 16SrIII, 16SrXIV, and 16SrXV), representing five new subgroups (16SrI-AO, 16SrXIV-D, 16SrXIV-E, 16SrXIV-F, and 16SrXV-C). The results strongly suggest that the newly identified phytoplasma strains not only represent new genetic subgroup lineages, but also extend previously undiscovered geographical distributions. In addition, ten phytoplasma-harboring leafhoppers belonged to seven known leafhopper species, none of which were previously reported insect vectors of phytoplasmas. The findings from this study provide fresh insight into genetic diversity, geographical distribution, and insect host range of phytoplasmas. Further transmission trials and screening of new potential host plants and weed reservoirs in areas adjacent to collection sites of phytoplasma harboring leafhoppers will contribute to a better understanding of phytoplasma transmission and epidemiology. 

Phytoplasmas are phloem-limited plant pathogenic bacteria in the class Mollicutes transmitted by sap-feeding insect vectors of the Order Hemiptera. Vectors still have not yet been identified for about half of the 33 known phytoplasma groups and this has greatly hindered efforts to control the spread of diseases affecting important crops. Extensive gaps of knowledge on actual phytoplasma vectors and on the plant disease epidemiology prevent our understanding of the basic underlying biological mechanisms that facilitate interactions between insects, phytoplasmas and their host plants. This paper presents a complete online database of Hemiptera-Phytoplasma-Plant (HPP) biological interactions worldwide, searchable via an online interface. The raw data are available through Zenodo at https://doi.org/10.5281/zenodo.2532738. The online database search interface was created using the 3I software (Dmitriev 2006) which enhances data usability by providing a customised web interface (http://trivellone.speciesfile.org/) that provides an overview of the recorded biological interactions and ability to discover particular interactions by searching for one or more phytoplasma, insect or plant taxa. The database will facilitate synthesis of all available and relevant data on the observed associations between phytoplasmas and their insect and plant hosts and will provide useful data to generate and test ecological and evolutionary hypotheses. 

Phytoplasmas are a diverse monophyletic group of phytopathogenic bacteria. No attempts have yet been made to the estimate the divergence times of phytoplasma lineages. Reltime molecular divergence time analyses using 16S rRNA gene sequence data of Acholeplasmataceae was performed. The timetree of phytoplasmas, provided here for the first time, and based on prior divergence time estimates for two nodes within the Mollicutes, estimates the split between phytoplasma and Acholeplasma at about 663 million years ago (Ma), and initial diversification of the crown phytoplasma clade at about 330 Ma. Overall, these results suggest that phytoplasmas have been associated with insect and plant hosts at least since the carboniferous period. 

Compilation and retrieval of reliable data on biological interactions is one of the critical bottlenecks affecting efficiency and statistical power in testing ecological theories. TaxonWorks, a web-based workbench, can facilitate such research by enabling the digitization of complex biological interactions involving multiple species, individuals, and trophic levels. These data can be further organized into spatial and temporal axes, and annotated at the level of individual or grouped interactions (e.g. singularly citing the combined elements of a tritrophic interaction). The simple, customizable nature of tools ultimately reduces the time-consuming steps of data gathering, cleaning, and formatting of datasets for subsequent exploration and analysis while also improving the asserted semantics. An example use case is provided with a dataset of associations among plants, pathogens and insect vectors. The curated data are accessed through the JSON serving TaxonWorks API (Application Programming Interface) by an R package. Analysis and visualization of the network graphs persisted in TaxonWorks is demonstrated using core R functionality and the igraph package (Csardi and Nepusz 2006). TaxonWorks is open-source, collaboratively built software available at http://taxonworks.org. 

Phytoplasmas (Mollicutes,Acholeplasmataceae), vector‐borne obligate bacterial plant parasites, infect nearly 1,000 plant species and unknown numbers of insects, mainly leafhoppers (Hemiptera, Deltocephalinae), which play a key role in transmission and epidemiology. Although the plant–phytoplasma–insect association has been evolving for >300 million years, nearly all known phytoplasmas have been discovered as a result of the damage inflicted by phytoplasma diseases on crops. Few efforts have been made to study phytoplasmas occurring in noneconomically important plants in natural habitats. In this study, a subsample of leafhopper specimens preserved in a large museum biorepository was analyzed to unveil potential new associations. PCR screening for phytoplasmas performed on 227 phloem‐feeding leafhoppers collected worldwide from natural habitats revealed the presence of 6 different previously unknown phytoplasma strains. This indicates that museum collections of herbivorous insects represent a rich and largely untapped resource for discovery of new plant pathogens, that natural areas worldwide harbor a diverse but largely undiscovered diversity of phytoplasmas and potential insect vectors, and that independent epidemiological cycles occur in such habitats, posing a potential threat of disease spillover into agricultural systems. Larger‐scale future investigations will contribute to a better understanding of phytoplasma genetic diversity, insect host range, and insect‐borne phytoplasma transmission and provide an early warning for the emergence of new phytoplasma diseases across global agroecosystems.