Chrysanthemum Yellows Phytoplasma Research Articles

Increased susceptibility to Chrysanthemum Yellows phytoplasma infection in Atcals7ko plants is accompanied by enhanced expression of carbohydrate transporters

Main conclusionLoss of CALS7 appears to confer increased susceptibility to phytoplasma infection in Arabidopsis, altering expression of genes involved in sugar metabolism and membrane transport.Callose deposition around sieve pores, under control of callose synthase 7 (CALS7), has been interpreted as a mechanical response to limit pathogen spread in phytoplasma-infected plants. Wild-type and Atcals7ko mutants were, therefore, employed to unveil the mode of involvement of CALS7 in the plant’s response to phytoplasma infection. The fresh weights of healthy and CY-(Chrysanthemum Yellows) phytoplasma-infected Arabidopsis wild type and mutant plants indicated two superimposed effects of the absence of CALS7: a partial impairment of photo-assimilate transport and a stimulated phytoplasma proliferation as illustrated by a significantly increased phytoplasma titre in Atcal7ko mutants. Further studies solely dealt with the effects of CALS7 absence on phytoplasma growth. Phytoplasma infection affected sieve-element substructure to a larger extent in mutants than in wild-type plants, which was also true for the levels of some free carbohydrates. Moreover, infection induced a similar upregulation of gene expression of enzymes involved in sucrose cleavage (AtSUS5, AtSUS6) and transmembrane transport (AtSWEET11) in mutants and wild-type plants, but an increased gene expression of carbohydrate transmembrane transporters (AtSWEET12, AtSTP13, AtSUC3) in infected mutants only. It remains still unclear how the absence of AtCALS7 leads to gene upregulation and how an increased intercellular mobility of carbohydrates and possibly effectors contributes to a higher susceptibility. It is also unclear if modified sieve-pore structures in mutants allow a better spread of phytoplasmas giving rise to higher titre.

Silencing of ATP synthase β reduces phytoplasma multiplication in a leafhopper vector

The leafhopper Euscelidius variegatus is a natural vector of the chrysanthemum yellows phytoplasma (CYp) and a laboratory vector of the Flavescence dorée phytoplasma (FDp). Previous studies indicated a crucial role for insect ATP synthase α and β subunits during phytoplasma infection of the vector species. Gene silencing of ATP synthase β was obtained by injection of specific dsRNAs in E. variegatus. Here we present the long-lasting nature of such silencing, its effects on the small RNA profile, the significant reduction of the corresponding protein expression, and the impact on phytoplasma acquisition capability. The specific transcript expression was silenced at least up to 37 days post injection with an average reduction of 100 times in insects injected with dsRNAs targeting ATP synthase β (dsATP) compared with those injected with dsRNAs targeting green fluorescent protein (dsGFP), used as negative controls. Specific silencing of this gene was also confirmed at protein level at 15 days after the injection. Total sRNA reads mapping to dsATP and dsGFP sequences in analysed libraries showed in both cases a peak of 21 nt, a length consistent with the generation of dsRNA-derived siRNAs by RNAi pathway. Reads mapped exclusively to the fragment corresponding to the injected dsATPs, probably indicating the absence of a secondary machinery for siRNA synthesis. Insects injected either with dsATP or dsGFP successfully acquired CYp and FDp during feeding on infected plants. However, the average phytoplasma amount in dsATP insects was significantly lower than that measured in dsGFP specimens, indicating a probable reduction of the pathogen multiplication when ATP synthase β was silenced. The role of the insect ATP synthase β during phytoplasma infection process is discussed.

RNA interference of muscle actin and ATP synthase beta increases mortality of the phytoplasma vector Euscelidius variegatus.

RNA interference (RNAi) techniques have emerged as powerful tools to develop novel management strategies for the control of insect pests. The leafhopper Euscelidius variegatus is a natural vector of chrysanthemum yellows phytoplasma and a laboratory vector of Flavescence dorée phytoplasma. Phytoplasmas are insect-borne bacterial plant pathogens that cause economically relevant crop losses worldwide. In this study, we demonstrated that microinjection of muscle actin and ATP synthase β double-stranded (ds)RNAs into adult insects caused an exponential reduction in the expression of both genes, which began within 72 h of dsRNA administration and lasted for 14 days, leading to almost complete silencing of the target genes. Such silencing effects on muscle actin expression appeared to be both time- and dose-dependent. Our results also showed that the knockdown of both genes caused a significant decrease in survival rates in comparison with green fluorescent protein (GFP) dsRNA-injected control insects. The effectiveness of RNAi-based gene silencing in E. variegatus guarantees the availability of a powerful reverse genetic tool for the functional annotation of its genes and the identification of those potentially involved in the interaction with phytoplasmas. In addition, this study demonstrated that muscle actin and ATP synthase β may represent candidate genes for RNAi-based control of E. variegatus. © 2018 Society of Chemical Industry.

Two Phytoplasmas Elicit Different Responses in the Insect Vector Euscelidius variegatus Kirschbaum.

Phytoplasmas are plant-pathogenic bacteria transmitted by hemipteran insects. The leafhopper Euscelidius variegatus is a natural vector of chrysanthemum yellows phytoplasma (CYp) and a laboratory vector of flavescence dorée phytoplasma (FDp). The two phytoplasmas induce different effects on this species: CYp slightly improves whereas FDp negatively affects insect fitness. To investigate the molecular bases of these different responses, transcriptome sequencing (RNA-seq) analysis of E. variegatus infected with either CYp or FDp was performed. The sequencing provided the first de novo transcriptome assembly for a phytoplasma vector and a starting point for further analyses on differentially regulated genes, mainly related to immune system and energy metabolism. Insect phenoloxidase activity, immunocompetence, and body pigmentation were measured to investigate the immune response, while respiration and movement rates were quantified to confirm the effects on energy metabolism. The activation of the insect immune response upon infection with FDp, which is not naturally transmitted by E. variegatus, confirmed that this bacterium is mostly perceived as a potential pathogen. Conversely, the acquisition of CYp, which is naturally transmitted by E. variegatus, seems to increase the insect fitness by inducing a prompt response to stress. This long-term relationship is likely to improve survival and dispersal of the infected insect, thus enhancing the opportunity of phytoplasma transmission.

Genome sequence, prevalence and quantification of the first iflavirus identified in a phytoplasma insect vector.

The leafhopper Euscelidius variegatus is a natural vector of chrysanthemum yellows phytoplasma (CY) and an efficient vector of flavescence dorée phytoplasma (FD) under laboratory conditions. During a transcriptome sequencing (RNA-seq) project aimed at investigating the interactions between the insect and the two phytoplasmas, a 10,616-nucleotide-long contig with high sequence similarity to known picorna-like viruses was identified among the assembled insect transcripts. The discovery came totally unexpected, because insects from the laboratory colony did not show any evident symptom that could be related to the presence of a virus. The amino acid sequence, the shape and size of viral particles, and the results of phylogenetic analysis suggest that this virus, named Euscelidius variegatus virus 1 (EVV-1), can be considered a new member of a new species in the genus Iflavirus. EVV-1 was detected in all of the tested insects from the laboratory colony used for RNA-seq, both in phytoplasma-exposed and in non-exposed insects, but the viral load measured in FD-exposed samples was significantly lower than that in non-exposed insects. This result suggests the possible existence of an intriguing cross-talk among insects, endogenous bacteria, and viruses. The identification of two other E. variegatus laboratory colonies that were free of EVV-1 could represent the key to addressing some basic virological issues, e.g., viral replication and transmission mechanisms, and offer the opportunity to use infectious clones to express heterologous genes in the leafhopper and manipulate the expression of endogenous genes by promoting virus-induced gene silencing.

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.

Effects of two AM fungi on phytoplasma infection in the model plant Chrysanthemum carinatum

Phytoplasmas are plant pathogenic bacteria, naturally transmitted by insects and confined in the phloem of the host plant, where they take up nutrients and eventually cause plant death. Their control is mainly based on insecticide treatments. The aim of this work was to study the effect of two AM fungi in modifying plant response to chrysanthemum yellows phytoplasma (CY) infection in chrysanthemum plants. Inoculation of Glomus intraradices BB-E and G. mosseae BEG12 reduced the damage caused by this plant pathogen in the aerial part of the plant, increased plant tolerance to the infection and reduced the severity of symptom expression, probably in a systemic way. Inoculation with G. mosseae did not alter CY multiplication and viability in young leaves, whilst the morphology of CY mature leaves was typical of senescent cells. Possible mechanisms involved are discussed.

Increased plant tolerance against chrysanthemum yellows phytoplasma (‘Candidatus Phytoplasma asteris’) following double inoculation with Glomus mosseae BEG12 and Pseudomonas putida S1Pf1Rif

The aim of this work was to assess the effects of a combined inoculum of a rhizobacterium and an arbuscular mycorrhizal (AM) fungus on plant responses to phytoplasma infection, and on phytoplasma multiplication and viability in Chrysanthemum carinatum plants infected by chrysanthemum yellows phytoplasma (CY). Combined inoculation with Glomus mosseae BEG12 and Pseudomonas putida S1Pf1Rif resulted in some resistance to phytoplasma infection (about 30%), delayed symptom expression in nonresistant plants, improved growth of the aerial part of the infected plants (+68·1%), and altered root morphology (root tip number: +49·9%; branching degree: +82·8%). Combined inoculation with the two beneficial microorganisms did not alter CY multiplication and viability. In inoculated and infected plants, phytoplasma morphology was typical of senescent cells. A more active and efficient root system in double‐inoculated plants probably mediated the effects of the two rhizospheric microorganisms in the infected plants. The practical application of rhizospheric microorganisms for mitigating phytoplasma damage, following evaluation under field conditions, represents an additional tool for the integrated management of phytoplasmosis.

Host Plant Determines the Phytoplasma Transmission Competence of Empoasca decipiens (Hemiptera: Cicadellidae)

Phytoplasmas are phloem-restricted plant pathogens transmitted by leafhoppers, planthoppers, and psyllids (Hemiptera). Most known phytoplasma vectors belong to the Cicadellidae, but many are still unknown. Within this family, Empoasca spp. (Typhlocybinae) have tested positive for the presence of some phytoplasmas, and phytoplasma transmission has been proven for one species. The aim of this work was to investigate the ability of Empoasca decipiens Paoli in transmitting chrysanthemum yellows phytoplasma (CYP, "Candidatus Phytoplasma asteris", 16SrI-B) and Flavescence dorée phytoplasma (FDP, 16SrV-C) to Chrysanthemum carinatum Schousboe (tricolor daisy) and Viciafaba (L.) (broad bean). Euscelidius variegatus Kirschbaum, a known vector of CYP and FDP, was caged together with Em. decipiens on the same source plants as a positive control of acquisition. Em. decipiens acquired CYP from daisies, but not from broad beans, and inoculated the pathogen to daisies with alow efficiency, but not to broad beans. Em. decipiens did not acquire FDP from the broad bean source. Consistent with the low transmission rate, CYP was found in the salivary glands of very few phytoplasma-infected Em. decipiens, indicating these organs represent a barrier to phytoplasma colonization. In the same experiments, the vector Eu. variegatus efficiently acquired both phytoplasmas, and consistently CYP was detected in the salivary glands of most samples of this species. The identity of the CYP strain in leafhoppers and plants was confirmed by polymerase chain reaction (PCR)-restriction fragment length polymorphism. The CYP titer in Em. decipiens was monitored over time by real-time PCR. The damage caused by Em. decipiens feeding punctures was depicted. Differences in feeding behavior on different plant species may explain the different phytoplasma transmission capability. Em. decipiens proved to be an experimental vector of CYP.

Effects of Pseudomonas putida S1Pf1Rif Against Chrysanthemum Yellows Phytoplasma Infection

Phytoplasmas cause damage on a number of plant species leading to relevant economical loss. Up to now, strategies to limit their spread led to only partial success. In this context, the use of plant-beneficial bacteria to control phytoplasmas has never been explored. The aim of this work was to assess the effect of Pseudomonas putida S1Pf1Rif against chrysanthemum yellows phytoplasma (CYP) infection of daisy. Plant biomass, root architecture, symptom severity, phytoplasma titer, and viability were evaluated in inoculated and control plants. CYP reduced plant growth and root development. Although the phytoplasma titer in young apical leaves was not affected by inoculation with S1Pf1Rif, the pseudomonad improved plant growth of CYP-infected plants. Whereas CYP titer increased over time in uninoculated plants, its viability decreased, regardless of the presence of P. putida S1Pf1Rif. Finally, phytoplasma cells in fully developed leaves of CYP-infected plants inoculated with S1Pf1Rif often appeared degenerated. Overall, our results indicate that P. putida S1Pf1Rif is able to alleviate the disease, although it does not affect the presence of viable phytoplasmas in young, developing leaves of the infected plants.

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.

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.

Quantification over time of chrysanthemum yellows phytoplasma (16Sr-I) in leaves and roots of the host plant Chrysanthemum carinatum (Schousboe) following inoculation with its insect vector

The rate of increase of chrysanthemum yellows phytoplasma (CYP, 16Sr-I) in leaves and roots of the host plant Chrysanthemum carinatum following localised and free insect inoculation is described. The fate of CYP in each test plant has been monitored over time using a relative quantification method based on real time PCR. Significant differences in CYP titre have been found both in apical leaves and roots sampled at different times post inoculation. CYP was much more concentrated in young apical leaves and roots compared to old basal leaves. Few thousands and few hundred thousands CYP genome units per nanogram of plant DNA were the maximum amounts found in leaves and roots, respectively. Active movement of CYP towards young developing leaves and roots has been demonstrated.

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.

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.

Differential acquisition of chrysanthemum yellows phytoplasma by three leafhopper species

AbstractChrysanthemum yellows (CY) phytoplasma has been transmitted with three leafhopper species: Euscelidius variegatus (Kirschbaum), Macrosteles quadripunctulatus (Kirschbaum) and Euscelis incisus (Kirschbaum): the first two species are reported as CY phytoplasma vectors for the first time. Leafhoppers were allowed to acquire the pathogen from the following source plants: Apium graveolens L., Catharanthus roseus L., Chrysanthemum carinatum Schousboe L. and C. frutescens L. DNA extracted from healthy or inoculative leafhoppers‐exposed plants were analyzed by dot‐blot and Southern hybridizations with a molecular probe constructed onto a fragment of European aster yellows phytoplasma DNA. The three leafhopper species were able to transmit CY phytoplasma after acquisition on chrysanthemum, but only M. quadripunctulatus and E. variegatus transmitted after feeding on periwinkle, and none acquired it from celery. All plant species tested were susceptible to CY, but while chrysanthemum and periwinkle were suitable for both inoculation and acquisition, celery did not seem to be a good source of phytoplasma for further inoculations. It is concluded that host plants influence leafhoppers' vectoring ability, possibly due to the different feeding behaviour of the insects on diverse plant species. Since CY, like several other phytoplasmas, can be transmitted by different insect species, it is likely that a close transmission specificity probably does not exist between phytoplasmas and their leafhopper vectors.