Multiplex PCR for Discriminating Host Plant Associations of Hyalesthes obsoletus (Hemiptera: Cixiidae), a Key Vector and Driver of 'Ca. Phytoplasma solani' Epidemiology

Given the ecological and epidemiological specialization of Hyalesthes obsoletus, the principle vector of ‘Candidatus Phytoplasma solani’, the primary objective of this study was to develop molecular tools for discriminating three host plant associations of the vector populations: (i) Convolvulus arvensis–Urtica dioica (Ca–Ud), (ii) Vitex agnus-castus (Vac), and (iii) Crepis foetida (Cf). The genetic diversity of the nearly full-length mitochondrial COI gene (1467 bp) was analyzed and compared among previously reported and newly collected individuals of the three host plant associations on a wide geographic range. Multiplex PCR was designed and evaluated for discriminating H. obsoletus host plant associations based on the size of amplified fragments: 1084 bp for the Cf association, 645 bp for the Ca–Ud association, and 355 bp for the Vac association. Examples of the epidemiological value of combining data on the genetic characteristics of the vector and the pathogen are provided. The method is intended to facilitate an accurate identification of the vector’s phylogenetic lineage, natural host plant preference, and epidemiological transmission routes of ‘Ca. P. solani’. When applied to H. obsoletus specimens collected from cultivated plants within an agroecosystem and combined with ‘Ca. P. solani’ genotyping, the method should provide valuable information on disease epidemiology, source(s) of emergence, and transmission routes

Case 3752 – Curculio antirrhini Paykull, 1800 (currently Rhinusa antirrhini; Insecta, Coleoptera, Curculionoidea, Curculionidae): proposed precedence over Curculio noctis Herbst, 1795

The purpose of this application, under Articles 23.9.3, 81.2.1 and 81.2.3 of the Code, is to conserve the name Curculio antirrhini Paykull, 1800, a common Palaearctic weevil species currently belonging to the genus Rhinusa Stephens, 1829 (Curculionoidea, Curculionidae) by giving it precedence over the little-used older name Curculio noctis Herbst, 1795 whenever these names are considered synonyms

Immature stages of Palearctic Mecinus species (Coleoptera, Curculionidae, Curculioninae): morphological characters diagnostic at genus and species levels

The immature stages of ten Mecinus species are described for the first time and those of two other species are redescribed, adding important chaetotaxy characters that were missing from previous descriptions. These species belong to six of the nine assemblages of Mecinus species previously established according to a phylogenetic analysis. All these groupings are confirmed on the basis of several characters of mature larvae and pupae. Moreover, all the species show several characters that are useful for distinguishing them from each other, including cryptic species that previously had few differential characters. Some characters that may be useful for separating Mecinus from other genera in the tribe are suggested. To confirm the taxonomic identification of some larvae, the mtCOII gene was obtained and compared with sequences from identified adult specimens. The most important characters for separating the immature stages of the genera and species groups in Mecinus are the number of palpomeres of the labial palpi (1 or 2), the number of air tubes of the thoracic and abdominal spiracles (unicameral or bicameral), and the number of epipharyngeal setae. The species studied herein were compared with those known from other genera in the tribe Mecinini. Two keys, one to the described larvae and the other to the pupae, are provided. Detailed biological data, several of which are new, on some species are reported

Description of immature stages of Gymnetron species (Coleoptera, Curculionidae, Curculioninae), with particular emphasis on the diagnostic morphological characters at the generic and specific levels

The immature stages of the following five Palaearctic Gymnetron species are described for the first time: G. tibiellum Desbrochers des Loges, 1900, G. veronicae (Germar, 1821), G. rotundicolle Gyllenhal, 1838, G. melanarium (Germar, 1821), and G. villosulum Gyllenhal, 1838. These species belong to four different groups previously established according to a phylogenetic analysis: the first two belong to the G. veronicae group and the other three to groups respectively bearing their name (G. rotundicolle, G. melanarium, and G. villosulum groups). All these species exhibit several diagnostic characters distinguishing them from each other. Some characters that can be used to separate Gymnetron from other genera in the tribe are also suggested. Three highly significant characters for the larvae and three for the pupae were identified. For the larvae they are: (1) labial palpi with single palpomeres, (2) all spiracles unicameral, and (3) epipharynx with a single pair of mes or none at all. For the pupae they are: (1) the pronotum with prominent pronotal protuberances, (2) abdominal segment VIII with a conical abdominal protuberance dorsally, and (3) very short or even reduced urogomphi. The species studied here are compared with those Gymnetron species already known and with other genera in the tribe Mecinini. Keys to the larvae and pupae described here are provided. All the characters used for identification are illustrated by photographs or drawings

First report of the exotic weevil Stenopelmus rufinasus (Coleoptera: Curculionidae) occurrence in Iran

The water fern weevil, Stenopelmus rufinasus Gyllenhal (Coleoptera: Curculionidae), is considered as the most important biological control agent of Azolla spp. in the world. Azolla spp. was introduced in Iran in 1986. In August 2017, two specimens of S. rufinasus were collected on Azolla spp. in waterways near Anzali lagoon and Rice Research Institute of Iran (RRII) in Guilan province for the first time. Identification was based on molecular sequencing. This is the first record of S. rufinasus from Iran

Developmental Biology and Identification of a Garden Pest, Otiorhynchus (Podoropelmus) smreczynskii Cmoluch, 1968 (Coleoptera, Curculionidae, Entiminae), with Comments on Its Origin and Distribution

The mature larva and pupa of Otiorhynchus smreczynskii are described and illustrated with complete chaetotaxy for the first time. Five larval instars and the factors for larval growth are determined, and the larval development of this species is comprehensively described. In order to confirm species affiliation, selected larvae were subjected to genetic analysis (mtCOI). Host plants and unique feeding signs of some Entiminae species are presented, and all available data on development are documented and interpreted. Additionally, the morphometry of 78 specimens (48 of O. smreczynskii and 30 of O. rotundus) was examined in order to verify the usefulness of morphological features in distinguishing both species. The female genitalia of both species are illustrated, described and compared with each other for the first time. Finally, the updated distribution of O. smreczynskii is given, and a possible origin of O. smreczynskii and O. rotundus is proposed

Epidemiological Role of Dictyophara europaea (Hemiptera: Dictyopharidae) in the Transmission of ‘Candidatus Phytoplasma solani’

Bois noir, an economically important disease of grapevine yellows that causes significant economic losses in wine production, is associated with ‘Candidatus Phytoplasma solani’ and transmitted to grapevines by cixiids Hyalesthes obsoletus and Reptalus panzeri. Polyphagous planthopper Dictyophara europaea, commonly found in natural habitats, harbors phytoplasmas from distinct groups and is an alternative vector in the open epidemiological cycles of the Flavescence dorée phytoplasma in grapevine in European vineyards. This study addresses the role of D. europaea in the transmission cycle(s) of ‘Ca. P. solani’ among wild habitats, natural reservoir plants, and the vineyard agroecosystem using MLSA and transmission trials with naturally infected adults to grapevine and Catharanthus roseus. The infection rates of D. europaea ranged from 7% to 13% in diverse locations, while reservoir herbaceous plants were infected in the amount of 29%. A total of 13 CaPsol MLSA genotypes were detected in D. europaea (7) and plants (8). Nine of them corresponded to previously identified genotypes. Two new genotypes were found in D. europaea (tuf-b1/S1/V14/Rqg50-sv1 and tuf-b1/S18/V14/Rqg50-sv1) and one in Convolvulus arvensis (tuf-b1/S1/V2-TA/Rqg31-sv1), whereas one was shared by two hosts, Crepis foetida and Daucus carota (tuf-b1/S1/V2-TA/STOL-sv1). Naturally infected D. europaea successfully transmitted the tuf-b1/S1/V2-TA/STOL type to five grapevines and six periwinkles, tuf-b1/S1/V2-TA/Rqg31 to one grapevine, and tuf-b1/S1/V2-TA/Rqg50 to one periwinkle, indicating that D. europaea is an intermediate vector in CaPsol epidemiological cycles

Retrospective use of integrative taxonomy in classical biological control: The unintentional introduction of the weevil Rhinusa dieckmanni to North America

A seed-feeding weevil introduced to North America (NA) as a biological control agent of the invasive toadflax Linaria dalmatica (L.) Mill., identified then as Gymnetron antirrhini “Dalmatian host race” and subsequently confirmed as established, was revealed through our study to be a separate species, i.e., Rhinusa dieckmanni (Behne) (Coleoptera: Curculionidae). This weevil species was presumed to be endemic in its native range, with a distribution restricted to Mount Rila in southwestern Bulgaria. We conducted a comprehensive study of seed-feeding weevils associated with L. dalmatica, L. dalmatica ssp. macedonica (Griseb.) D.A. Sutton, L. genistifolia (L.) Mill., and L. grandiflora Desf. across a broad geographic area of their native range. Those results revealed that all four host plants were used by R. dieckmanni and thus the native geographic range of the species is wider than expected, encompassing the Balkans and the Anatolian Plateau. Our observations suggest that phenotypes of this weevil are highly variable and dependent on the seed capsule size of the Linaria host population. The haplotype network based on mitochondrial COII, 16S genes, and nuclear EF 1-α gene genealogy confirmed the conspecific nature of geographically distant weevil populations, that is, R. dieckmanni phenotypes utilizing L. genistifolia, L. dalmatica, and L. grandiflora for larval development. Specimens collected from L. dalmatica in the northwestern USA shared the same haplotypes as samples from L. dalmatica ssp. macedonica in southwestern North Macedonia, supporting the known introduction history of the North American population. Females from these populations have relatively short rostrums, which may limit their reproductive success on North American invasive L. dalmatica with larger seed capsules

Genetic Diversity of Flavescence Dorée Phytoplasmas in Vineyards of Serbia: From the Widespread Occurrence of Autochthonous Map-M51 to the Emergence of Endemic Map-FD2 (Vectotype II) and New Map-FD3 (Vectotype III) Epidemic Genotypes

Flavescence dorée (FD) is a European quarantine disease of grapevine caused by FD phytoplasma (FDp) transmitted by the leafhopper of North American origin Scaphoideus titanus. The disease affects the most important viticultural regions of Europe and all wine-growing regions of Serbia. Unlike the insect vector, the pathogen is native to Europe and associated with several wild host plants among which alder trees as the main source of two out of three map genetic clusters of pathogen variants (Map-FD1 and FD2). Heretofore, the FDp epidemic in Serbian vineyards was thought to be monotypic, i.e., caused by the single genotype of the Map-FD3 cluster, M51, and correlated with clematis as the natural source plant. This study aimed to provide data on genetic diversity, through map and vmpA gene typing, and insights into ecological properties of epidemiological cycles driving the epidemic outbreaks of FD in Serbia today. Map genotyping of 270 grapevine isolates collected from 2017 to 2019 confirmed M51 as autochthonous genotype widespread in all wine producing regions of Serbia and the dominant FDp epidemic genotype in most of the districts (75%, 202/270 isolates), except in north Serbia where multiple outbreaks of M12 Map-FD3 were recorded (54 isolates). Tree of heaven is reported as a new FDp plant reservoir for the Serbian vineyards, hosting the M51 genotype, along with clematis. An outbreak of a new endemic Map-FD3 genotype M144 was documented in grapevine samples from east Serbia (5 isolates), correlating with previous findings of the same genotype in clematis. In addition, single grapevine infections with five new Map-FD3 genotypes (M150-M154) were recorded in central Serbia, thus indicating high endemic potential for new outbreaks. The vmpA typing placed all Map-FD3 isolates into the VmpA-III cluster, i.e., Vectotype III. Finally, we found direct evidence that at least two FDp endemic genotypes, M89 and M148, of the Map-FD2/VmpA-II have escaped from alders and propagated in the grapevine-S. titanus pathosystem in Serbia (Vectotype II). Our findings confirm the high complexity of the FDp ecological cycle and provide evidence of a unique, autochthonous Balkan epidemiology sourced endemically

The “code red” for Balkan vineyards: occurrence of Orientus ishidae (Matsumura, 1902) (Hemiptera: Cicadellidae) in Serbia

Orientus ishidae (Matsumura, 1902) (Hemiptera: Cicadellidae), known as the mosaic leafhopper, is an invasive alien species native to the Eastern Palaearctic, but also widespread and well established on the eastern territory of North America. Since its first detection in Europe in 1998, this polyphagous species has expanded its distribution area and rapidly spread through central and western European countries, inhabiting a wide range of broadleaf trees and shrubs (e.g. Gleditsia triacanthos, Salix spp., Corylus spp., Acer spp., Betula spp., Populus spp. and Carpinus spp.), which are usually present in the surrounding of vineyards. Over the last decade, O. ishidae was frequently found within European vineyards and was proven to be able to complete its life cycle on grapevine. Moreover, this leafhopper was shown to be naturally infected with the Flavescence dorée phytoplasma (FDp), a devastating disease which causes significant economic losses in the major vine-producing countries. This study provides data on the occurrence of O. ishidae in Serbia, clearly confirming that it has expanded its distribution range to the Balkan Peninsula which could lead to establishment of new FDp epidemiological cycles inside the local vineyard regions. As a consequence, there could be an increased negative impact on phytosanitary situation with a continual spread of this vector species and FDp epidemic outbreaks in Southeastern Europe