Macrosteles Quadripunctulatus Research Articles

Evidence suggesting interactions between immunodominant membrane protein Imp of Flavescence dorée phytoplasma and protein extracts from distantly related insect species.

In this study, binding between the immunodominant membrane protein Imp of the 16SrV-D phytoplasma associated with Flavescence dorée disease (FD-Dp) and insect proteins of vectors and non-vectors of FD-Dp was tested. Six Auchenorrhyncha species, from distantly related groups were selected: Scaphoideus titanus, Euscelidius variegatus, Macrosteles quadripunctulatus, Zyginidia pullula (Cicadomorpha), Ricania speculum and Metcalfa pruinosa (Fulgoromorpha). The vector status of each species was retrieved from the literature or determined by transmission trials in this study. A His-tagged partial Imp protein and a rabbit polyclonal antibody were synthesized and used for Western and Far-Western dot Blot (FWdB) experiments. Total native and membrane proteins (MP) were extracted from entire bodies and organs (gut and salivary glands) of each insect species. FWdB showed decreasing interaction intensities of Imp fusion protein with total proteins from entire bodies of S. titanus, E. variegatus (competent vectors) and M. quadripunctulatus (non-vector), while no interaction signal was detected with the other three species (non-vectors). A strong signal detected upon interaction of FD-D Imp and MP from guts of closely related insects supports the role of this organ as the first barrier to ensure successful transmission. Our results showed that specific Imp binding, correlated with vector status, is involved in interactions between FD-Dp and insect proteins. Integrating knowledge on host-pathogen protein-protein interactions and on insect phylogeny would help to identify the actual range of vectors of phytoplasma strains of economic importance.

Phytoplasma Transmission: Insect Rearing and Infection Protocols.

Phytoplasmas are obligate pathogens and thus they can be studied only in association with their plants or insect hosts. In this chapter, we present protocols for rearing some phytoplasma insect vectors, to obtain infected insects and plants under controlled environmental conditions. We focus on Euscelidius variegatus and Macrosteles quadripunctulatus that can infect Arabidopsis thaliana, and Hyalesthes obsoletus and Scaphoideus titanus, that can infect grapevine.

Complete Genome Sequences of the Obligate Symbionts "Candidatus Sulcia muelleri" and "Ca. Nasuia deltocephalinicola" from the Pestiferous Leafhopper Macrosteles quadripunctulatus (Hemiptera: Cicadellidae).

Two bacterial symbionts of the European pest leafhopper, Macrosteles quadripunctulatus (Hemiptera: Cicadellidae), were fully sequenced. “Candidatus Sulcia muelleri” and “Ca. Nasuia deltocephalinicola” represent two of the smallest known bacterial genomes at 190 kb and 112 kb, respectively. Genome sequences are nearly identical to strains reported from the closely related host species, M. quadrilineatus.

Plant-mediated interspecific horizontal transmission of an intracellular symbiont in insects.

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.

Role of the major antigenic membrane protein in phytoplasma transmission by two insect vector species.

BackgroundPhytoplasmas are bacterial plant pathogens (class Mollicutes), transmitted by phloem feeding leafhoppers, planthoppers and psyllids in a persistent/propagative manner. Transmission of phytoplasmas is under the control of behavioral, environmental and geographical factors, but molecular interactions between membrane proteins of phytoplasma and vectors may also be involved. The aim of the work was to provide experimental evidence that in vivo interaction between phytoplasma antigenic membrane protein (Amp) and vector proteins has a role in the transmission process. In doing so, we also investigated the topology of the interaction at the gut epithelium and at the salivary glands, the two barriers encountered by the phytoplasma during vector colonization.MethodsExperiments were performed on the ‘Candidatus Phytoplasma asteris’ chrysanthemum yellows strain (CYP), and the two leafhopper vectors Macrosteles quadripunctulatus Kirschbaum and Euscelidius variegatus Kirschbaum.To specifically address the interaction of CYP Amp at the gut epithelium barrier, insects were artificially fed with media containing either the recombinant phytoplasma protein Amp, or the antibody (A416) or both, and transmission, acquisition and inoculation efficiencies were measured.An abdominal microinjection protocol was employed to specifically address the interaction of CYP Amp at the salivary gland barrier. Phytoplasma suspension was added with Amp or A416 or both, injected into healthy E. variegatus adults and then infection and inoculation efficiencies were measured.An internalization assay was developed, consisting of dissected salivary glands from healthy E. variegatus exposed to phytoplasma suspension alone or together with A416 antibody. The organs were then either observed in confocal microscopy or subjected to DNA extraction and phytoplasma quantification by qPCR, to visualize and quantify possible differences among treatments in localization/presence/number of CYP cells.ResultsArtificial feeding and abdominal microinjection protocols were developed to address the two barriers separately. The in vivo interactions between Amp of ‘Candidatus Phytoplasma asteris’ Chrysanthemum yellows strain (CYP) and vector proteins were studied by evaluating their effects on phytoplasma transmission by Euscelidius variegatus and Macrosteles quadripunctulatus leafhoppers. An internalization assay was developed, consisting of dissected salivary glands from healthy E. variegatus exposed to phytoplasma suspension alone or together with anti-Amp antibody. To visualize possible differences among treatments in localization/presence of CYP cells, the organs were observed in confocal microscopy. Pre-feeding of E. variegatus and M. quadripunctulatus on anti-Amp antibody resulted in a significant decrease of acquisition efficiencies in both species. Inoculation efficiency of microinjected E. variegatus with CYP suspension and anti-Amp antibody was significantly reduced compared to that of the control with phytoplasma suspension only. The possibility that this was due to reduced infection efficiency or antibody-mediated inhibition of phytoplasma multiplication was ruled out. These results provided the first indirect proof of the role of Amp in the transmission process.ConclusionProtocols were developed to assess the in vivo role of the phytoplasma native major antigenic membrane protein in two phases of the vector transmission process: movement through the midgut epithelium and colonization of the salivary glands. These methods will be useful also to characterize other phytoplasma-vector combinations. Results indicated for the first time that native CYP Amp is involved in vivo in specific crossing of the gut epithelium and salivary gland colonization during early phases of vector infection.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0522-5) contains supplementary material, which is available to authorized users.

Temperature-dependent transmission of Candidatus phytoplasma asteris by the vector leafhopper Macrosteles quadripunctulatus Kirschbaum

A set of experiments was carried out to characterize how temperature affects the spread of chrysanthemum yellows phytoplasma (CYP), a strain of Candidatus Phytoplasma asteris, in Chrysanthemum carinatum plants transmitted by the Macrosteles quadripunctulatus leafhopper. Experiments provided data on CYP latency period in insect and plant host, M. quadripunctulatus adult mortality rate, and epidemics progression in plants under controlled conditions inside climatic chambers. Experiments were conducted at temperatures ranging between 15 and 30°C. Empirical laws for temperature-dependent epidemiological parameters were next derived and used in a dynamical model of the epidemics progression. Experiments showed that CYP epidemics was faster at higher temperatures and the model could replicate these observations with relatively high accuracy (correlation >98.03% and residuals <14.5%). The epidemics spreading rate increased linearly from 0.2 plants infected per day at 15°C to about 0.7 plants per day at 30°C, possibly due to: i) faster CYP multiplication in the host plants and ii) higher frequency of feeding bouts of M. quadripunctulatus at higher temperatures.

Selection of reference genes from two leafhopper species challenged by phytoplasma infection, for gene expression studies by RT-qPCR

BackgroundPhytoplasmas are phloem-limited phytopathogenic wall-less bacteria and represent a major threat to agriculture worldwide. They are transmitted in a persistent, propagative manner by phloem-sucking Hemipteran insects. For gene expression studies based on mRNA quantification by RT-qPCR, stability of housekeeping genes is crucial. The aim of this study was the identification of reference genes to study the effect of phytoplasma infection on gene expression of two leafhopper vector species. The identified reference genes will be useful tools to investigate differential gene expression of leafhopper vectors upon phytoplasma infection.ResultsThe expression profiles of ribosomal 18S, actin, ATP synthase β, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and tropomyosin were determined in two leafhopper vector species (Hemiptera: Cicadellidae), both healthy and infected by “Candidatus Phytoplasma asteris” (chrysanthemum yellows phytoplasma strain, CYP). Insects were analyzed at three different times post acquisition, and expression stabilities of the selected genes were evaluated with BestKeeper, geNorm and Normfinder algorithms. In Euscelidius variegatus, all genes under all treatments were stable and could serve as reference genes. In Macrosteles quadripunctulatus, BestKeeper and Normfinder analysis indicated ATP synthase β, tropomyosin and GAPDH as the most stable, whereas geNorm identified reliable genes only for early stages of infection.ConclusionsIn this study a validation of five candidate reference genes was performed with three algorithms, and housekeeping genes were identified for over time transcript profiling of two leafhopper vector species infected by CYP. This work set up an experimental system to study the molecular basis of phytoplasma multiplication in the insect body, in order to elucidate mechanisms of vector specificity. Most of the sequences provided in this study are new for leafhoppers, which are vectors of economically important plant pathogens. Phylogenetic indications were also drawn from sequence analysis of these genes.

Determination and population fluctuations of Cicadellidae (Hemiptera: Cicadomorpha) species in pomegranate orchards in Aydin Province, Turkey

Many Cicadellidae (Hemiptera: Cicadomorpha) species may reach high populations in fruit orchards. Cicadellids can cause damage not only by sucking plant sap, but also by transmitting detrimental plant pathogens, which may become more important in many cases. It was aimed to determine Cicadellidae species and population fluctuations of common species in pomegranate orchards in Aydin (Nazilli, Karacasu) province in Turkey. Studies were conducted in two pomegranate orchards through 2009-2010. Samplings were done with two yellow sticky traps and by sweep net with 100-sweep-collection on weeds in each orchard. Additionally, cicadellids were collected by shaking one branch of each tree and 20 trees in total in each orchard. Cicadellidae species and their populations were therefore investigated both on trees and weeds in orchards. As a result, 19 cicadellid species were found in orchards. Of these species, Docotettix cornutus Ribaut, Fieberiella anategea Meyer-Arndt, Asymmetrasca decedens (Paoli)+Empoasca decipiens Paoli and Zyginidia pullula (Boheman) were common on the trees and A. decedens (Paoli)+E. decipiens Paoli, Cicadulina bipunctella (Matsumura), Macrosteles quadripunctulatus (Kirschbaum) were common on the weeds in the orchards. Fieberiella anategea and D. cornutus populations reached high levels in JulyNovember on yellow sticky traps in 2009, but remain low during 2010. Asymmetresca decedens + E. decipiens populations reached high levels in May-June and October in 2009 and 2010. On the other hand, Z. pullula population stayed at a very low level in the years 2009 and 2010.

Are traditional neem extract preparations as efficient as a commercial formulation of azadirachtin A?

Neem seeds contain many substances with insecticidal properties, the main insecticidal ingredient being azadirachtin A. In developing countries such as Mali, a neem seed water extract is prepared by soaking ground seeds in water for three or seven days. The aim of this study was to check the effectiveness of this extract in terms of azadirachtin A extraction yield and insecticidal activity. The yield of extraction was 0.19 g azadirachtin A/100 g seeds. The concentration of azadirachtin A in the seed extract was approximately 200 mg l−1, eight times higher than the recommended concentration of commercial products (25 mg l−1). A comparison of the extractive capacity of different solvents indicated that the best solvents were water and methanol. The azadirachtin A concentration declined in extracts stored for more than 3 days at a temperatures higher than 30 °C. Bioassays were performed on target insects (the leafhopper Macrosteles quadripunctulatus, the moth Spodoptera littoralis and the tobacco whitefly Bemisia tabaci) in order to compare the insecticidal activity of the neem extract with that of the commercial product Neemazal T/S and of a solution of pure azadirachtin A. The bioassays conducted on the leafhopper and the moth demonstrated that the neem extract at the recommended concentration (25 mg l−1 active ingredient) was as effective as the azadirachtin-based commercial product at the same concentration, while for the control of the whitefly B. tabaci a higher concentration of the water extract was needed.

Variation in vector competency depends on chrysanthemum yellows phytoplasma distribution within Euscelidius variegatus

AbstractPhytoplasmas 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.

Characterization of putative membrane protein genes of the ‘Candidatus Phytoplasma asteris’, chrysanthemum yellows isolate

To characterize potentially important surface-exposed proteins of the phytoplasma causing chrysanthemum yellows (CY), new primers were designed based on the conserved regions of 3 membrane protein genes of the completely sequenced onion yellows and aster yellows witches' broom phytoplasmas and were used to amplify CY DNA. The CY genes secY, amp, and artI, encoding the protein translocase subunit SecY, the antigenic membrane protein Amp and the arginine transporter ArtI, respectively, were cloned and completely sequenced. Alignment of CY-specific secY sequences with the corresponding genes of other phytoplasmas confirmed the 16S rDNA-based classification, while amp sequences were highly variable within the 'Candidatus Phytoplasma asteris'. Five CY partial sequences were cloned into the pRSetC expression vector, and 3 of the encoded protein fragments (Amp 64/651, Amp 64/224, ArtI 131/512) were expressed as fusion antigens for the production of CY-specific polyclonal antibodies (A416 against Amp 64/224; A407 against ArtI 131/512). A416 recognized, in Western blots, the full-length Amp from CY-infected plants (periwinkle, daisy) and insect vectors (Euscelidius variegatus, Macrosteles quadripunctulatus). A416 also reacted to European aster yellows, to primula yellows phytoplasmas, to northern Italian strains of 'Ca. Phytoplasma asteris' from lettuce and gladiolus, but it did not react to American aster yellows phytoplasma.

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

AbstractIn 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.

Interrelationships Between “<I>Candidatus</I> Phytoplasma asteris” and Its Leafhopper Vectors (Homoptera: Cicadellidae)

The titer of chrysanthemum yellows phytoplasma (CYP, "Candidatus Phytoplasma asteris") in the three vector species Euscelis incisus Kirschbaum, Euscelidius variegatus Kirschbaum, and Macrosteles quadripunctulatus Kirschbaum (Homoptera: Cicadellidae) was measured after controlled acquisition from infected Chrysanthemum carinatum (Schousboe) (daisy) plants. Phytoplasma DNA was quantified in relation to insect DNA (genome units [GU] of phytoplasma DNA per ng of insect DNA) by using a quantitative real-time polymerase chain reaction (PCR) procedure. The increase in phytoplasma titer recorded in hoppers after they were transferred to plants that were nonhosts for CYP provides definitive evidence for phytoplasma multiplication in leafhoppers. CYP multiplication over time in M. quadripunctulatus was much faster than in E. incisus and E. variegatus. CYP titer was also highest in M. quadripunctulatus, and this was reflected in the latent period in the insect. The mean latent period of CYP in M. quadripunctulatus was 18 d versus 30 d in E. variegatus. M. quadripunctulatus was the most efficient vector, giving 100% transmission for single insects compared with 75-82% for E. incisus or E. variegatus, respectively. By sequential transmission, we analyzed the time course of transmission: E. variegatus were persistently infective for life or until shortly before death. Occasionally, leafhoppers failed to maintain continuity of infectivity even after completion of the latent period. PCR analysis of transmitter and nontransmitter E. variegatus adults showed that some nontransmitters were CYP positive, whereas others were CYP negative. These findings suggest that both midgut and salivary gland barriers play a role in transmission efficiency.

Activity of some insecticides in preventing transmission of chrysanthemum yellows phytoplasma (‘ Candidatus Phytoplasma asteris’) by the leafhopper Macrosteles quadripunctulatus Kirschbaum

Four insecticides, the organophosphates (OPs) fenitrothion, chlorpyrifos ethyl and malathion, and the neonicotinoid imidacloprid, were tested for their capacity to limit the transmission of chrysanthemum yellows phytoplasma (CYP) by the leafhopper vector Macrosteles quadripunctulatus to potted daisy plants, Chrysanthemum carinatum. The OPs were applied to the foliage as sprays, while imidacloprid was applied to the soil as a drench. Transmission trials were first done with groups of 5 infective leafhoppers caged on single plants at 1, 4, 7, 10, 15 and 20 days after insecticide treatments. In a second experiment, 20 infective leafhoppers were released in a cage containing 32 treated daisies and were free to move among the plants as long as they lived. The influence of source plant treatments on phytoplasma acquisition by the vector was also investigated by caging leafhopper nymphs on treated plants; 3 weeks later, surviving leafhoppers were tested for the presence of CYP by polymerase chain reaction and by transmission assays. The first and second experiments, with acquisition access periods on untreated source plants, were designed to mimic primary infections due to leafhoppers coming from outside the field. The third experiment was designed to model secondary, within-crop spread of phytoplasmas, since the source plants were treated, in an attempt to prevent phytoplasma acquisition. The OP-treated plants were significantly less infected than water-treated plants only at 1 and 4 days after treatment application, while those treated with imidacloprid were significantly protected at all times tested. The second experiment confirmed that imidacloprid was much more effective than OPs in preventing the CYP transmission. When caged on source plants treated 40 days earlier, most leafhoppers died but the survivors were able to acquire and transmit the phytoplasma. We conclude that, when the primary purpose is to protect the plants from the incoming infectious insects, use of the neonicotinoid is advisable. When the primary purpose is to suppress the vector population, OPs are likely to be effective.

Relative Quantification of Chrysanthemum Yellows (16Sr I) Phytoplasma in Its Plant and Insect Host Using Real-Time Polymerase Chain Reaction

A real-time polymerase chain reaction (PCR) method for the quantification of chrysanthemum yellows (CY) phytoplasma DNA in its plant (Chrysanthemum carinatum) and insect (Macrosteles quadripunctulatus) host is described. The quantity of CY DNA was measured in each run relative to the amount of host DNA in the sample. Primers and a TaqMan probe for the specific PCR amplification of phytoplasma DNA were designed on a cloned CY-specific ribosomal fragment. Primers and TaqMan probes were also designed on sequences of the internal transcribed spacer region of the insect's ITS1 rDNA and of the plant's 18S rDNA for amplification from C. carinatum and M. quadripunculatus, respectively. Absolute quantification of CY DNA was achieved by comparison with a dilution series of the plasmid containing a CY 16S rDNA target sequence. Absolute quantification of plant and insect DNAs was achieved by comparison with a dilution series of the corresponding DNAs. Quantification of CY DNA in relation to host DNA was finally expressed as genome units (GU) of phytoplasma DNA per nanogram of host DNA. Relative quantification avoided influences due to different yields during the DNA extraction procedure. The quantity of CY DNA was about 10,000-20,000 GU/ng of plant DNA and about 30,000-50,000 GU/ng of insect DNA. The method described could be used to phytoplasma multiplication and movement in different plant and insect hosts.

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

SummarySpecies 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.

TRANSMISSION OF CHRYSANTHEMUM YELLOWS, A SUBGROUP 16SrI-B PHYTOPLASMA, TO GRAPEVINE BY FOUR LEAFHOPPER SPECIES

SUMMARY Chrysanthemum yellows phytoplasma (CY), subgroup 16SrI-B, was experimentally transmitted to seedlings of grapevine (Vitis vinifera), cv. ‘Barbera’ and ‘White Muscat’ with four leafhopper species: Euscelidius variegatus, Euscelis incisus, Macrosteles quadripunctulatus and Scaphoideus titanus. Acquisition was accomplished in three days by 3 rd instar nymphs on CY-infected marguerites (Chrysanthemum carinatum). After three weeks, insects were moved to new test plants of the same species and allowed to feed for seven days. They were then transferred to grapevine seedlings for another seven-day-period, and so on as long as they survived. The four leafhopper species transmitted CY to both marguerite and grapevine with the following respective rates: M. quadripunctulatus, 100 (25/25) and 33.3 (9/27); E. variegatus , 59.6 (31/52) and 47.5 (28/59); E. incisus, 95.6 (22/23) and 33.3 (6/18); S. titanus 12.5 (7/56) and 17.0% (8/47). Symptoms on C. carinatum were typical of those already described, while on V. vinifera they consisted of growth reduction and abnormal production of axillary shoots bearing small chlorotic leaves. Downward rolling and reddening of basal leaves was occasionally observed on cv. ‘Barbera’. RFLP analysis of ribosomal DNA amplified in direct and/or nested PCRs driven by universal or 16SrI group-specific primers confirmed that the phytoplasma present in symptomatic plants, either used as inoculum or obtained by transmission, and in inoculative S. titanus did belong to subgroup 16SrI-B and was identical to the CY reference isolate.

Vector‐pathogen‐host plant relationships of chrysanthemum yellows (CY) phytoplasma and the vector leafhoppers Macrosteles quadripunctulatus and Euscelidius variegatus

AbstractChrysanthemum yellows (CY) phytoplasma is a plant‐pathogenic mollicutes belonging to the 16Sr‐IB genetic group which infects a variety of dicotyledonous plants and is transmitted in nature by some species of Cicadellidae Deltocephalinae. The transmission characteristics of CY and the factors influencing the vector efficiencies of the leafhoppers Macrosteles quadripunctulatus Kirschbaum and Euscelidius variegatus Kirschbaum are described in the present study using transmission experiments and phytoplasma‐specific polymerase chain reaction (PCR) assays. Vector insects were allowed to acquire CY under different experimental conditions and then transferred to healthy test plants for inoculation and/or sampled for DNA extraction and amplification. The transmission efficiency of CY was very high and almost all the leafhoppers became infective following acquisition on CY‐infected daisies. The latent period in the vector ranged from 16 to 20 days after the start of the acquisition and infectivity lasted, in general, for life. The PCR assay was successful in detecting CY phytoplasmas in the insects well before they became infective (5 versus 16–18 days) and was used to estimate the proportion of infective insects. When analysed for CY presence by PCR, all the leafhoppers fed for 7–18 days on source daisy reacted positively while, following one day of acquisition, some insects failed to provide amplification. Host‐plant species influenced CY acquisition, and daisy appeared a more efficient source for both leafhoppers compared to periwinkle. Life stage did not appear to be critical for CY acquisition, although newly‐hatched nymphs of E. variegatus acquired CY less efficiently than fifth instar nymphs.

Monitoring phytoplasma‐bearing leafhoppers/planthoppers in vineyards in the Golan Heights, Israel

In the course of identifying the vector(s) of the grapevine yellows (GY) and western‐X (WX) phytoplasmas in the Golan Heights, leafhopper and planthopper species were examined. The planthopper, Hyalesthes obsoletus, was trapped on yellow sticky traps or collected on weeds; there was a relatively small peak during two weeks in June and during four weeks starting mid‐September. The leafhoppers, Neoaliturus spp. and Circulifer sp. (of the haematoceps complex) were both trapped early or very late in summer, but could be collected on weeds throughout the summer. The two remaining leafhopper species, Macrosteles quadripunctulatus and Orosius orientalis (=albicinctus) were only rarely caught on sticky traps, but were found on weeds throughout the summer. Phytoplasmas were found within the body of these five species; all are historically known to be efficient vectors of various phytoplasmas and were therefore chosen for further investigations.

Cyclamen (Cyclamen persicum L.): a dead-end host species for 16Sr-IB and -IC subgroup phytoplasmas

Phytoplasmas belonging to the 16S rDNA subgroups IB and IC were found in five cyclamen (Cyclamen persicum L.) plants showing virescence and yellow stunted leaves and one plant showing phyllody, rolled and thickened leaves, respectively. Two cyclamens, representing the two syndromes, were chosen as source plants for transmission trials in which three leafhopper species, known as vectors of IB and IC subgroup phytoplasmas, were used to inoculate cyclamen and periwinkle [Catharanthus roseus (L.) G. Don] test plants. Out of 366 tested plants only one periwinkle exposed to Euscelis incisus was found harbouring a 16Sr-IB phytoplasma. Out of 60 tested vector insects, only one adult of Macrosteles quadripunctulatus and two of E. incisus fed on 16Sr-IB source cyclamen gave a positive amplification signal in nested PCR. This extremely low level of transmission to both cyclamen and the very susceptible periwinkle strongly suggests that cyclamen, commonly found infected in crops, is an unsuitable species for phytoplasma acquisition and can be regarded as a dead-end host plant for phytoplasmas belonging to both IB and IC subgroups. Indications for glasshouse management are drawn from these findings. Among the leafhoppers investigated E. incisus falls most under suspicion since it feeds better than the others on cyclamen, was able to transmit the disease to one periwinkle plant, and IB phytoplasmas were detected in two individuals.