Investigation on ‘bois noir’ epidemiology in north‐eastern Italian vineyards through a multidisciplinary approach

A multidisciplinary approach, based on field surveys, molecular biology techniques, and spatial data analyses, was utilised to investigate the Bois noir (BN) epidemiology in north‐eastern Italian vineyards during the years 2010–12. Symptomatic grapevines, weeds and specimens of the insect vector Hyalesthes obsoletus were monitored and mapped. Leaf samples from symptomatic grapevines and weeds, and captured insect specimens were analyzed by real‐time PCR to identify BN phytoplasma (BNp; ‘Candidatus Phytoplasma solani’ species), the etiological agent of BN. Data spatial distribution was analyzed using SADIE (Spatial Analysis by Distance IndicEs). Bois noir phytoplasma strains identified in weed candidates for an epidemiological role were characterised by RFLP‐based analyses of tuf gene amplicons. Results highlighted that, in the examined areas, the host systems Convolvulus arvensis – H. obsoletus and Urtica dioica – H. obsoletus play the main role in BN diffusion. It was also evidenced that other weeds (i.e. Chenopodium album and Malva sylvestris) spatially associated with symptomatic grapevines and/or insect vectors and infected by the same tuf type identified in grapevines and insects, could play a role in BN diffusion. On the other hand, some weeds (i.e. Trifolium repens) were uninfected and not associated with symptomatic grapevines and/or insect vectors. The synergic application of our multidisciplinary approach improved the knowledge of BN epidemiology, and provided helpful indication for designing experimental plans to contain BN spreading in vineyards through weed management. The approach described in the present work could be used to investigate the complex epidemiology of other phytoplasma diseases.     

Vitex agnus‐castus cannot be used as trap plant for the vector Hyalesthes obsoletus to prevent infections by ‘Candidatus Phytoplasma solani’ in northern Italian vineyards: Experimental evidence

Bois noir (BN), the most prevalent disease of the grapevine yellows complex, causes considerable yield loss in vineyards. BN is associated with phytoplasma strains of the species ‘Candidatus Phytoplasma solani’ (taxonomic subgroup 16SrXII‐A). In Europe, the BN phytoplasma is transmitted to grapevine mainly by Hyalesthes obsoletus, a polyphagous cixiid completing its life cycle on stinging nettle and field bindweed. As a result of the complexity of BN epidemiology, no effective control strategies have been developed. In previous studies conducted in the eastern Mediterranean coast of Israel, chaste tree (Vitex agnus‐castus) was found to be the preferred host plant of H. obsoletus but did not harbour BN phytoplasma. Thus, a ‘push and pull’ strategy was suggested based on the fact that chaste tree plants located at vineyard borders was an effective trap plant for H. obsoletus adults. However, in other studies carried out in the eastern Adriatic coast of Montenegro, chaste tree was found to be a key source plant for BN phytoplasma transmission to grapevine. This study aimed to investigate (i) the interaction between chaste tree and H. obsoletus through survival, attractiveness and oviposition experiments conducted comparing the behaviour of H. obsoletus in chaste tree versus stinging nettle and grapevine and (ii) the capability of chaste tree to harbor ‘Ca. P. solani’ in northern Italy through transmission trials. H. obsoletus adults were found to survive on chaste tree and grapevine over a 1 week period and prefer chaste tree to grapevine. Moreover, H. obsoletus produced eggs and overwintered as nymphs on chaste tree, even if at a lesser extent than on stinging nettle. H. obsoletus originating from nettle was found able to transmit ‘Ca. P. solani’ to chaste tree (2 plants of 16 were found infected by the BN phytoplasma strain St5 identified in H. obsoletus specimens). These results increased our knowledge about the role of Vitex agnus‐castus as host plant of H. obsoletus and BN phytoplasma in northern Italy and do not recommend considering chaste tree as trap plant at vineyard borders.     

Population structure and incidence of the stolbur phytoplasma vector Hyalesthes obsoletus (Cixiidae) among geographic regions in Switzerland

The dissemination of stolbur phytoplasma (16Sr‐XIIA group)‐induced yellows diseases depends on the dispersal biology and host plant fidelity of the planthopper vector Hyalesthes obsoletus (Hemiptera: Cixiidae). We analysed the degree of these two properties in H. obsoletus by studying its population genetic structure and stolbur infection rates relative to the two major host plants, Convolvolus arvensis and Urtica dioica, in order to infer relevant divisions for stolbur epidemiology in Swiss viticultural regions. Three regional populations with the potential to determine stolbur epidemiology in distinct ways were identified. First, populations associated with U. dioica in northern Switzerland were most related to genetically distinct U. dioica host race populations identified previously in Germany. Second, populations in central and southwest Switzerland were undifferentiated relative to host plant and likely have wider stolbur transmission breadths than the northern specialized populations. Third, populations in south of the Alps (Ticino) were undifferentiated relative to host plant but geographically isolated from other Swiss regions, thus implying separate population dynamics in this area. The knowledge of these three distinct epidemiological cycles will help to adapt management programmes against stolbur diseases in Swiss vineyards.     

Infection of Bois‐Noir tuf‐type‐I stolbur phytoplasma in Hyalesthes obsoletus (Hemiptera: Cixiidae) larvae and influence on larval size

Recent dramatic spread of the grapevine yellows disease Bois Noir (BN) in Germany is above all explained by highly increased abundances of the vector Hyalesthes obsoletus (Hemiptera: Cixiidae) associated to the plant Urtica dioica, the reservoir of the BN pathogen stolbur tuf‐type‐I. The vector acquires BN‐phytoplasma as larvae whilst feeding on the roots of infected U. dioica. To understand the dynamics of the Urtica‐cycle, we tested at what instar larvae become infected and whether infection affects larvae size (i.e. growth) at two sites in the Mosel Valley, Germany. Larvae were tested from infected plants and collected at instar‐stages 3, 4 and 5. Larvae at stage 3 were already infected but infection rates increased significantly between stage 3 and 5, mean infection rates: 0.12–0.62. There was no effect of infection on larval size at any instar stage.     

Host plant preferences of Hyalesthes obsoletus,the vector of the grapevine yellows disease ‘bois noir’, in Switzerland

Bois noir is an important grapevine yellows disease in Europe that can cause serious economic losses in grapevine production. It is caused by stolbur phytoplasma strains of the taxonomic group 16Sr‐XII‐A. Hyalesthes obsoletus Signoret (Hemiptera: Cixiidae) is the most important vector of bois noir in Europe. This polyphagous planthopper is assumed to mainly use stinging nettle [Urtica dioica L. (Urticaceae)] and field bindweed [Convolvulus arvensis L. (Convolvulaceae)] as its host plants. For a better understanding of the epidemiology of bois noir in Switzerland, host plant preferences of H. obsoletus were studied in the field and in the laboratory. In vineyards of Western Switzerland, adults of H. obsoletus were primarily captured on U. dioica, but a few specimens were also caught on C. arvensis, hedge bindweed [Calystegia sepium (L.) R. Brown (Convolvulaceae)], and five other dicotyledons [i.e., Clematis vitalba L. (Ranunculaceae), Lepidium draba L. (Brassicaceae), Plantago lanceolata L. (Plantaginaceae), Polygonum aviculare L. (Polygonaceae), and Taraxacum officinale Weber (Asteraceae)]. The preference of the vector for U. dioica compared to C. arvensis was confirmed by a second, more targeted field study and by the positioning of emergence traps above the two plant species. Two‐choice experiments in the laboratory showed that H. obsoletus adults originating from U. dioica preferred to feed and to oviposit on U. dioica compared to C. arvensis. However, H. obsoletus nymphs showed no host plant preference, even though they developed much better on U. dioica than on C. arvensis. Similarly, adults survived significantly longer on U. dioica than on C. arvensis or any other plant species tested [i.e., L. draba and Lavandula angustifolia Mill. (Lamiaceae)]. In conclusion, although nymphs of H. obsoletus had no inherent host plant preference, adults tested preferred to feed and oviposit on U. dioica, which is in agreement with the observed superior performance of both nymphal and adult stages on this plant species. Urtica dioica appears to be the principal host plant of H. obsoletus in Switzerland and plays therefore an important role in the epidemiology of the bois noir disease in Swiss vineyards.     

Controlling ‘bois noir’ disease on grapevine: does the timing of herbicide application affect vector emergence?

Bois noir is an important grapevine yellows disease that can cause serious economical losses in European grapevine production. Hyalesthes obsoletus Signoret (Hemiptera, Cixiidae) is the principal vector of bois noir in Switzerland and stinging nettle (Urtica dioica) is its favourite host plant species in vineyards. As bois noir disease can hardly be cured and direct control measures against H. obsoletus are ineffective, viticultural control practices target stinging nettle, the actual reservoir and source of both the pathogen and its vector. Currently, it is recommended to apply herbicides against stinging nettle at the end of the season to kill developing H. obsoletus nymphs. To verify if this late period of herbicide application is justified, stinging nettle patches were treated with glyphosate in the autumn, in the spring or were left untreated as a control. Herbicide applications at both dates controlled the growth of stinging nettle very well in the subsequent summer, although the autumnal treatment was slightly more efficient. To study glyphosate’s direct impact on the development of H. obsoletus nymphs, emergence traps were placed directly in the centre of treated and untreated stinging nettle patches. There was no significant difference among the three treatments in the total number of adults emerging. Thus, an aerial application of glyphosate in either spring or autumn did not inhibit the nymphs’ development on the roots of stinging nettle in the soil. Our results challenge current recommendations of applying herbicides against stinging nettle at the end of the season and suggest that stinging nettle could also be controlled in spring, alike other viticultural weeds.     

Entomopathogenic nematodes and fungi to control Hyalesthes obsoletus (Hemiptera: Auchenorrhyncha: Cixiidae)

Hyalesthes obsoletus Signoret (Hemiptera: Auchenorrhyncha: Cixiidae) is a univoltine, polyphagous planthopper that completes its life cycle, including the subterranean nymph cryptic stage, on herbaceous weeds. In vineyards, it can transmit ‘Candidatus Phytoplasma solani’, an obligate parasitic bacterium associated with bois noir (BN) disease of grapevine, from its host plants to grapevine when occasionally feeding on the latter. The main disease management strategies are based on vector(s) control. Insecticide treatments on grapevine canopy are completely inefficient on H. obsoletus, due to its life cycle. Consequently, control of this planthopper focuses on the nymphs living on the roots of their host plants. Such practices, based on herbicide application and/or weed management, can reduce vector density in the vineyard but can impact the environment or may not be applicable, highlighting the necessity for alternative strategies. In this study, the efficacy of entomopathogenic nematodes (EPNs; Steinernema carpocapsae, S. feltiae, Heterorhabditis bacteriophora) and fungi (EPFs; Beauveria bassiana, Metarhizium anisopliae, Isaria fumosorosea, Lecanicillium muscarium) against H. obsoletus nymphs (EPNs) and adults (EPNs and EPFs) was assessed under laboratory and greenhouse conditions. The majority of examined EPNs and EPFs were able to kill H. obsoletus exhibiting a range of effectiveness. S. carpocapsae (among EPNs) and I. fumosorosea (among EPFs) were found to be the most effective biocontrol agents in all trials carried out. Advantages and limitations of such promising biocontrol agents were discussed. Ecological competency and conditions that can impede or enhance the EPNs and EPFs performance should be investigated to optimize their performance under field conditions.

     

Vector activity of Hyalesthes obsoletus living on nettles and transmitting a stolbur phytoplasma to grapevines: a case study

We report a case study on the vector activity of a Hyalesthes obsoletus (Hemiptera: Cixiidae) population living on nettle plants (Urtica dioica) and transmitting a stolbur phytoplasma (Sp) to grapevines (Vitis vinifera). The research was conducted in a site that included a vineyard bordered with a large fallow area where nettles were the predominant plant species together with sparse old grapevines. Nettles hosted a high population of H. obsoletus. By using transparent sticky traps to sample adults, we observed that the daily flight activity of males and females to grapevines in the fallow was unimodal peaking between 15 and 21 h in the day. Adults were unable of great dispersion into the vineyard and the pattern of insect captures inside the planting reflected the pattern of Sp‐infected grapevines in the late autumn. When insects were forced to feed on grapevine cuttings for transmission assays, survival of H. obsoletus decreased after 24–48 h. The scarce propensity of the vector to move into the vineyard and feed on grapevines was counterbalanced by the rapidity of H. obsoletus to inoculate Sp to grapevines (estimated minimum inoculation access period ranged from 3 to 6 h) and a relative high incidence of Sp in the population of H. obsoletus that ranged between 20% and 30% of sampled insects as shown by a polymerase chain reaction–based procedure. Characterisation of Sp by restriction fragment length polymorphism analysis of nonribosomal phytoplasma DNA showed the occurrence of an Sp strain known to infect H. obsoletus associated to nettles and grapevines in Germany.     

Spatial dispersion patterns of potential leafhopper and planthopper (Homoptera) vectors of phytoplasma in wine vineyards

Species from three subfamilies of Cicadellidae and one species of Cixiidae, all potential vectors of phytoplasmas, were trapped in vineyards over the course of two years. These insects were caught on yellow sticky traps during the spring; virtually none were trapped during the summer months. Molecular analysis (PCR) of selected samples of the leafhoppers and planthoppers for presence of phytoplasma DNA was conducted on most species. Neoaliturus fenestratus was the most abundant known vector species and was positive for stolbur (Stol) and aster yellows (AY) phytoplasma. Circulifer haematoceps complex, which were also positive for Stol and AY, were about 10‐fold fewer than N. fenestratus. Hyalesthes obsoletus, also a known phytoplasma vector, occurred in substantial numbers only late in the season when the vines and leaves were dehiscing and turning brown, but was positive for Stol and AY. A species whose vector status is unknown (Megophthalmus scabripennis) was also caught in large numbers and was shown, by PCR analysis, to be positive for AY. Other known vector species trapped included: Anaceratagallia laevis (positive for AY), Austroagallia sinuata, Dryodurades sp. (dlabolai?) (positive for AY), Macrosteles quadripunctulatus (positive for Stol), and Orosius orientalis. The spatial dispersion pattern of the four most abundant species were investigated by using Moran's spatial statistic; N. fenestratus and M. scabripennis showed significant spatial patterns, whereas C. haematoceps and H. obsoletus did not.     

Invasion biology and host specificity of the grapevine yellows disease vector Hyalesthes obsoletus in Europe

Within the past 10 years, the yellows disease ‘bois noir’ (BN) has become one of the commercially most important diseases of grapevine [Vitis vinifera L. (Vitaceae)] in Europe. Infection pressure is caused by phytoplasmas of the stolbur 16SrXII‐A group that are transmitted by a planthopper vector, Hyalesthes obsoletus Signoret (Homoptera: Auchenorrhyncha). Infestation happens as an accidental side‐effect of the feeding behaviour of the vector, as vector and pathogen proliferation is dependent on other plants. In Germany, the increase of BN is correlated with the use of a new host plant by the vector, increase in abundance of the vector on the new host plant, and dissemination of host plant‐specific pathogen strains. In this article, we investigate geographic and host‐associated range expansion of the vector. We test whether host‐plant utilization in Germany, hence the increase in BN, is related to genetic host races of the vector and, if so, whether these have evolved locally or have immigrated from southern populations that traditionally use the new host plant. The genetic population analysis demonstrates a recent expansion and circum‐alpine invasion of H. obsoletus into German and northern French wine‐growing regions, which coincides with the emergence of BN. No H. obsoletus mitochondrial DNA haplotype host‐plant affiliation was found, implying that the ability to use alternative host plants is genetically intrinsic to H. obsoletus. However, subtle yet significant random amplified polymorphic DNA (RAPD) genetic differentiation was found among host plant populations. When combined, these results suggest that a geographic range expansion of H. obsoletus only partly explains the increase of BN, and that interactions with host plants also occur. Further possible beneficial factors to H. obsoletus, such as temperature increase and phytoplasma interactions, are discussed.     

Universal and group-specific real-time PCR diagnosis of flavescence dorée (16Sr-V), bois noir (16Sr-XII) and apple proliferation (16Sr-X) phytoplasmas from field-collected plant hosts and insect vectors

Three real‐time PCR–based assays for the specific diagnosis of flavescence dorée (FD), bois noir (BN) and apple proliferation (AP) phytoplasmas and a universal one for the detection of phytoplasmas belonging to groups 16Sr‐V, 16Sr‐X and 16Sr‐XII have been developed. Ribosomal‐based primers CYS2Fw/Rv and TaqMan probe CYS2 were used for universal diagnosis in real‐time PCR. For group‐specific detection of FD phytoplasma, ribosomal‐based primers fAY/rEY, specific for 16Sr‐V phytoplasmas, were chosen. For diagnosis of BN and AP phytoplasmas, specific primers were designed on non‐ribosomal and nitroreductase DNA sequences, respectively. SYBR^® Green I detection coupled with melting curve analysis was used in each group‐specific protocol. Field‐collected grapevines infected with FD and BN phytoplasmas and apple trees infected with AP phytoplasma, together with Scaphoideus titanus, Hyalesthes obsoletus and Cacopsylla melanoneura adults, captured in the same vineyards and orchards, were used as templates in real‐time PCR assays. The diagnostic efficiency of each group‐specific protocol was compared with well‐established detection procedures, based on conventional nested PCR. Universal amplification was obtained in real‐time PCR from DNAs of European aster yellows (16Sr‐I), elm yellows (16Sr‐V), stolbur (16Sr‐XII) and AP phytoplasma reference isolates maintained in periwinkles. The same assay detected phytoplasma DNA in all test plants and test insect vectors infected with FD, BN and AP phytoplasmas. Our group‐specific assays detected FD, BN, and AP phytoplasmas with high efficiencies, similar to those obtained with nested PCR and did not amplify phytoplasma DNA of other taxonomic groups. Melting curve analysis was necessary for the correct identification of the specific amplicons generated in the presence of very low target concentrations. Our work shows that real‐time PCR methods can sensitively and rapidly detect phytoplasmas at the universal or group‐specific level. This should be useful in developing defence strategies and for quantitative studies of phytoplasma–plant–vector interactions.     

Origin of a sudden mass occurrence of the stolbur phytoplasma vector Hyalesthes obsoletus (Cixiidae) in Austria

The grapevine disease ‘bois noir’ is widespread in European viticulture, but in many regions there is a lack of correspondence between disease spread and abundance of the main insect vector, the planthopper Hyalesthes obsoletus. This was the situation in Austria until 2012, when a mass occurrence of the vector was observed on Urtica dioica, a new host plant for the vector and reservoir plant for the pathogen, stolbur phytoplasma, in this area. Here we analyse the origin of the Austrian vector populations using genetic markers. The origin was unambiguously assigned to two regional populations, and two causes for the population expansion: immigration of East Central European populations and local demographic expansion. The observed population increase was thus independent of phylogenetic ancestry, but linked to the host plant and the exchange of a specific stolbur phytoplasma strain between the two vector populations. These circumstances are identical to but independent of the emergence of bois noir west of the European Alps, where an exchange between other vector populations associated with U. dioica of another stolbur phytoplasma genotype has led to disease outbreaks. Combined, the independent outbreaks in Austria and Europe west of the Alps are suggestive of an active role for stolbur phytoplasma in the vector–plant interaction and thus the host distribution of the vector.     

Molecular phylogeny of Trissolcus species (Hymenoptera: Scelionidae)

Trissolcus species (Hymenoptera: Scelionidae) are the most promising biological control agents against sunn pest. The accurate identification of natural enemies is crucial in order to develop successful biological control programs. This paper presents phylogenetic analyses of Trissolcus species based on data sets consisting of 18S, 28S, ITS1, ITS2 and cytochrome oxidase subunit I (COI) genes. The most commonly used genetic loci in Trissolcus species identification is the cytochrome oxidase I gene (COI). Also restriction fragment length polymorphism analyses of PCR amplified COI gene have been developed to discriminate closely related species. We suggest that Trissolcus grandis Thompson and Trissolcus semistriatus Nees split significantly into two different genetic groups while there is another species diverging from T. semistriatus which is decsribed as ‘Trissolcus flavotibialis Kocak & Guz, n. sp.’     

Study of the transmission of stolbur phytoplasma to different crop species, by Macrosteles quadripunctulatus

In an epidemiological study conducted on commercial agricultural plots affected by stolbur phytoplasma in Northern and Central Spain, different species of leafhoppers and planthoppers were identified as potential vectors of the phytoplasma. They included individuals of Macrosteles quadripunctulatus infected by stolbur phtytoplasma in most of the locations. The potential of this species as a vector of stolbur was evaluated in this work. The transmission trials were carried out on healthy plants of Catharanthus roseus (periwinkle), Lycopersicon esculentum (tomato), Daucus carota (carrot), Lactuca sativa (lettuce) and Vitis vinifera (grapevine). The first symptoms of infection in these plants were observed 2 weeks after the inoculation period in tomato and periwinkle, and after 4 weeks in carrot. Only one of five grapevines showed phytoplasma symptoms. PCR analysis was used to verify the ability of M. quadripunctulatus in transmitting stolbur phytoplasma in the plant species tested. The phytoplasma was not detected in lettuce or in the healthy control plants. Studies of stolbur transmission to insect‐feeding medium were also conducted and indicated that M. quadripunctulatus acquires and was capable of transmitting the phytoplasma after it fed during a single day on infected plants followed by a 19‐day latent period on healthy plants.     

Developing a new molecular marker for aphid species identification: Evaluation of eleven candidate genes with species-level sampling

The mitochondrial cytochrome c oxidase subunit I (COI) gene has been utilized as a molecular marker for aphid species identification. However, this gene has sometimes resulted in misidentification because of low interspecific genetic divergences between some species pairs. In this study, to propose new molecular markers for the family Aphididae, we first screened 2289 sequences of 11 genes (COI, COII, CytB, ATP6, lrRNA, srRNA, ITS1, ITS2, EF1a, 18S, and 28S) collected from the GenBank. Among the 11 genes, ATP6 gene revealed the largest genetic divergence among congeneric species with the smallest divergence among conspecific individuals; in contrast, species pairs with low genetic divergences (< 1%) were not observed. Secondly, for statistically testing the usefulness of ATP6 gene in species identification, we analyzed genetic distances between all of the combinations of 32 individuals of 20 species for both COI and ATP6 genes. The ATP6 gene showed lower intraspecific (on average 0.08%) and higher interspecific (on average 8.28%) genetic distances than the COI gene (on average 0.19% and 6.24%, respectively) for the same pairs of individuals. This study corroborates the usefulness of the ATP6 gene as a new molecular marker that could improve the misidentification problems that are inherent with the COI gene.     

Survival relative to new and ancestral host plants,phytoplasma infection,and genetic constitution in host races of a polyphagous insect disease vector

Dissemination of vectorborne diseases depends strongly on the vector's host range and the pathogen's reservoir range. Because vectors interact with pathogens, the direction and strength of a vector's host shift is vital for understanding epidemiology and is embedded in the framework of ecological specialization. This study investigates survival in host‐race evolution of a polyphagous insect disease vector, Hyalesthes obsoletus, whether survival is related to the direction of the host shift (from field bindweed to stinging nettle), the interaction with plant‐specific strains of obligate vectored pathogens/symbionts (stolbur phytoplasma), and whether survival is related to genetic differentiation between the host races. We used a twice repeated, identical nested experimental design to study survival of the vector on alternative hosts and relative to infection status. Survival was tested with Kaplan–Meier analyses, while genetic differentiation between vector populations was quantified with microsatellite allele frequencies. We found significant direct effects of host plant (reduced survival on wrong hosts) and sex (males survive longer than females) in both host races and relative effects of host (nettle animals more affected than bindweed animals) and sex (males more affected than females). Survival of bindweed animals was significantly higher on symptomatic than nonsymptomatic field bindweed, but in the second experiment only. Infection potentially had a positive effect on survival in nettle animals but due to low infection rates the results remain suggestive. Genetic differentiation was not related to survival. Greater negative plant‐transfer effect but no negative effect of stolbur in the derived host race suggests preadaptation to the new pathogen/symbiont strain before strong diversifying selection during the specialization process. Physiological maladaptation or failure to accept the ancestral plant will have similar consequences, namely positive assortative mating within host races and a reduction in the likelihood of oviposition on the alternative plant and thus the acquisition of alternative stolbur strains.     

Impact of Vector Dispersal and Host-Plant Fidelity on the Dissemination of an Emerging Plant Pathogen

Dissemination of vector-transmitted pathogens depend on the survival and dispersal of the vector and the vector''s ability to transmit the pathogen, while the host range of vector and pathogen determine the breath of transmission possibilities. In this study, we address how the interaction between dispersal and plant fidelities of a pathogen (stolbur phytoplasma tuf-a) and its vector (Hyalesthes obsoletus: Cixiidae) affect the emergence of the pathogen. Using genetic markers, we analysed the geographic origin and range expansion of both organisms in Western Europe and, specifically, whether the pathogen''s dissemination in the northern range is caused by resident vectors widening their host-plant use from field bindweed to stinging nettle, and subsequent host specialisation. We found evidence for common origins of pathogen and vector south of the European Alps. Genetic patterns in vector populations show signals of secondary range expansion in Western Europe leading to dissemination of tuf-a pathogens, which might be newly acquired and of hybrid origin. Hence, the emergence of stolbur tuf-a in the northern range was explained by secondary immigration of vectors carrying stinging nettle-specialised tuf-a, not by widening the host-plant spectrum of resident vectors with pathogen transmission from field bindweed to stinging nettle nor by primary co-migration from the resident vector''s historical area of origin. The introduction of tuf-a to stinging nettle in the northern range was therefore independent of vector''s host-plant specialisation but the rapid pathogen dissemination depended on the vector''s host shift, whereas the general dissemination elsewhere was linked to plant specialisation of the pathogen but not of the vector.     

Molecular identification and phylogenetic analysis of spider mites (Prostigmata: Tetranychidae) of Turkey

The family Tetranychidae includes many agriculturally important species known as spider mites. Their morphological identification is quite difficult due to the tiny size of their taxonomic characters and the requirement for high-level expertise. This may lead to pest misidentification and thus failure in pest management. DNA-based species identification seems to offer an alternative solution to overcome these issues. In the present study, two common molecular markers—Cytochrome oxidase subunit I (COI) and Internal transcribed spacer 2 (ITS2)—were used to identify 10 spider mite species from Turkey. Furthermore, genetic distances for several of them were assessed. Panonychus ulmi and Bryobia kissophila had the lowest (1.1%) and highest (4.5%) intra-specific genetic distances, respectively. In addition, integrative taxonomy allowed to identify Eotetranychus quercicola in Turkey as a new record. The sequences herein obtained will allow rapid species identification using molecular techniques and will contribute to resolve the phylogenetic history of spider mites.

     

Molecular identification of four Panonychus species (Acari: Tetranychidae) in Korea,including new records of P. caglei and P. mori

Four species of the genus Panonychus are identified in Korea including two previously known species P. citri (McGregor) and P. ulmi (Koch) and two newly identified species P. mori Yokoyama and P. caglei Mellot. Morphological diagnostics were observed among the four species in the color of dorsal tubercles and in the shape of male aedeagus. The dorsal tubercles of P. citri, P. ulmi, P. mori and P. caglei are red, white, whitish red and light red, respectively. The aedeagi of male adults are diagnostic in sigmoid‐shaped at the distal end and its shape and length was different among four species. Molecular comparison of the internal transcribed spacer 2 (ITS2) sequence of nuclear DNA and cytochrome‐c oxidase subunit I (COI) nucleotide sequence of mitochondrial DNA among the four species showed divergences 8–12% and 9–12%, respectively. Molecular analysis of the ITS2 and COI sequences revealed their divergences were slightly different among four species. In addition, species‐specific primer sets were designed at the base on ITS2 sequences to precisely diagnose these four species at the molecular level.