Effect of two strains of Flavescence dorée phytoplasma on the survival and fecundity of the experimental leafhopper vector Euscelidius variegatus Kirschbaum

We have investigated the influence on longevity and fecundity of Flavescence dorée phytoplasma (FDP), the agent of a grapevine yellows disease, in the experimental vector Euscelidius variegatus Kirschbaum. Late instar nymphs were exposed to one or the other of two strains of FDP (FD92 and FD2000) by feeding on infected broad bean (Vicia faba L.) or on healthy broad bean or maize (Zea mays L.) for an acquisition access period of 13 days. Detection of FDP in individual insects was done with PCR assays and revealed that almost all exposed leafhoppers had acquired FDP, for both FD92 and FD2000 strains. FDP infection significantly reduced the life span of males and females (ANOVA of the quartiles of survival distribution and Weibull scale parameter). FDP-exposed females produced significantly fewer nymphs. The two FDP strains had similar effects on reduction of survival and fecundity of leafhoppers. There was no significant differences in longevity of E. variegatus males exposed to FD broad bean than held on healthy broad bean or maize, but female survival and fecundity were reduced when they fed on maize versus healthy broad bean.     

Patterns of phytoplasma-infected and infective Scaphoideus titanus leafhoppers in vineyards with high incidence of Flavescence dorée

The incidence and transmissibility of Flavescence dorée phytoplasma (FDP) in populations of the vector Scaphoideus titanus Ball (Homoptera: Cicadellidae) were investigated by periodically collecting nymphs and adults of the leafhopper species in four vineyards with high incidence of Flavescence dorée (FD)‐diseased grapevines. Insects were tested individually for FDP with an ELISA procedure, after transmission assays to broadbean seedlings and further transmission to grapevine cuttings. No transmission occurred when early or middle instar nymphs were used to inoculate broadbeans, although a limited number of fifth‐instar nymphs and young adults transmitted the pathogen to broadbean seedlings. However, the same batches of insects transmitted FDP more efficiently to grapevine cuttings during prolonged inoculation periods, confirming the existence of a latent period before infected insects become infective. The proportions of ELISA‐positive individuals in the three categories of insects used for transmission assays reflected the rate of FDP transmission to grapevine cuttings. According to the data obtained by ELISA and from field sampling of first‐instar nymphs, we adapted the proportions of nymph hatching, of infected leafhoppers, and of infective leafhoppers (assuming a conservative latent period in the vector of 30 days) to logistic models as a function of degree‐days. We then discussed the possible use of the model developed for improving vector control decisions in FD‐infected vineyards.     

Survival,fecundity, and body mass of Amplicephalus curtulus influenced by ‘Candidatus Phytoplasma ulmi’ (16SrV‐A) infection

The leafhopper Amplicephalus curtulus Linnavuori & DeLong (Hemiptera: Cicadellidae) can transmit ‘Candidatus Phytoplasma ulmi’ (16SrV‐A) from a native Chilean shrub, Ugni molinae Turcz. (Myrtaceae), to ryegrasses. A recent study showed that this phytoplasma reduced the total protein content and the activity of detoxifying enzymes in A. curtulus, which could also affect its vector fitness. This study evaluated the effect of ‘Ca. Phytoplasma ulmi’ on the longevity, fecundity, and body mass of A. curtulus. Both females and males were exposed to ‘Ca. Phytoplasma ulmi’‐infected plants for 96 h, whereas a control group remained unexposed. Quartiles from adult emergence to 75% (t₇₅), 50% (t₅₀), and 25% (t₂₅) survival rates were determined for each leafhopper survival distribution. The dry weight was also established at the end of the experiment. The adult lifespan of phytoplasma‐infected males and females was significantly lower than that of the uninfected leafhoppers in quartile survival distributions t₅₀ and t₂₅. The phytoplasma‐infected males and females lived 3 and 4 weeks less than uninfected ones in the last quartile, respectively. Fecundity was established by number of nymphs per female (in four periods) in phytoplasma‐infected and uninfected assays. In general, the weekly pattern of the number of nymphs per phytoplasma‐infected female was lower than that of uninfected leafhoppers; it was 37% lower at the end of the experiment. Phytoplasma‐infected females weighed significantly less (11%) than uninfected individuals. Phytoplasma‐infected males weighed 8% less than uninfected ones, but this difference was not significant. Our data indicated that ‘Ca. Phytoplasma ulmi’ negatively affected the fitness of A. curtulus, and nymphs produced by phytoplasma‐infected females varied over time, which may influence the disease dynamics in nature or in field crops.     

New insights on Flavescence dorée phytoplasma ecology in the vineyard agro‐ecosystem in southern Switzerland

Phytoplasmas associated with Flavescence dorée (FDp) grapevine disease are quarantine pathogens controlled through mandatory measures including the prompt eradication and destruction of diseased plants, and the insecticide treatments against the insect vector, the ampelophagous leafhopper Scaphoideus titanus. In the present study, a multidisciplinary approach has been applied to investigate the FDp ecological cycle in a test vineyard agro‐ecosystem in Canton Ticino, south Switzerland. Despite the scarce population density of S. titanus, a regular trend of new infections (3.4% of the total vines) through the years was observed. The leafhopper Orientus ishidae was found as the most abundant among the captured insect species known as phytoplasma vectors (245 out of 315 specimens). The population of O. ishidae was evidenced prevalently (167 specimens) in the south‐western side of the vineyard and within the neighbouring forest constituted mainly by hazel (Corylus avellana) and willow (Salix spp.). These plant species were found infected by FDp related strains (30% of analysed trees) for the first time in this study. Interestingly, O. ishidae was found to harbour FDp related strains in high percentage (26% of the analysed pools). In addition, 16SrV phytoplasma group was detected for the first time in the insect Hyalesthes obsoletus and a FDp related strain in Thamnotettix dilutior, present in low populations within the test vineyard. Molecular characterisation and phylogenetic analyses of methionine aminopeptidase (map) gene sequences of FDp and related strains, here identified, revealed the great prevalence of the map‐type FD2 in grapevines (97%) and in O. ishidae pools (72%). Such a map‐type was found also in hazel and in T. dilutior, but not in S. titanus. Moreover, map‐types FD1 and FD3 were identified for the first time in Switzerland in several host plants and phytoplasma vectors, including grapevine (FD1), S. titanus (FD1) and O. ishidae (FD1 and FD3). Based on the data obtained in this study, it is reasonable to hypothesise that the ecological cycle of FDp could be related not exclusively to the grapevine‐specific feeding diet of S. titanus, but it could include other insect vector(s) and/or plant host(s). Further studies will be needed to prove the role of O. ishidae as vector able to transmit FDp from wild plants (e.g. hazel) to grapevine.     

First Detection of Stolbur Phytoplasma in Grapevines (Vitis vinifera cv. Merlot) Affected with Grapevine Yellows in Bulgaria

Between 2003 and 2005, a survey was conducted throughout the grape‐growing regions of Bulgaria to identify possible infection with grapevine yellows diseases, especially Flavescence dorée (FD). The samples were checked for phytoplasmas and viruses inducing similar symptoms in the Central Laboratory for Plant Quarantine. To confirm stolbur phytoplasma infection of grapevine, a multiplex nested‐PCR assay for direct detection of FD and stolbur phytoplasmas was used. Infection of grapevine with phytoplasma was detected. The disease is very common disease in Bulgaria on tomatoes, potatoes and other crops. Monitoring is being continued. This is the first report of phytoplasma‐infected grapevine in Bulgaria.     

An 1-Aminocyclopropane-1-carboxylate (ACC) deaminase-expressing endophyte increases plant resistance to flavescence dorée phytoplasma infection

Flavescence dorée is an epidemic yellows disease of grapevine, caused by a phytoplasma (FDP), for which there is currently no cure. We assessed whether the endophyte Pseudomonas migulae 8R6, able to synthesize 1-aminocyclopropane-1-carboxylate (ACC) deaminase, can limit the phytoplasma-induced damages in periwinkle, a model plant hosting FDP. Plant protection induced by 8R6 and its mutant, impaired in ACC deaminase synthesis, was compared. Fifteen plants per treatment were used; FD infection was transmitted by grafting. Evaluation of symptoms was performed every 4 days for 40 days. The presence and the amount of FDP were assessed by nested PCR and qPCR, respectively. Images of phytoplasma inside the infected plants were obtained by transmission electron microscopy. The strain 8R6 significantly reduced the number of symptomatic plants (53% vs 93%). While the density of FDP inside the leaves was unaffected by the bacterial strains, the FDP titre was under the quantification threshold in 38% of the plants inoculated with strain 8R6. Microscopical observations showed damaged FDP cells in plants inoculated with strain 8R6. The ACC deaminase activity of the endophytic bacteria P. migulae 8R6 helps the plant to regulate the level of the stress-related hormone ethylene, leading to significantly improved resistance to phytoplasma infection.     

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.     

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.     

Mixed Infection and Natural Spread of ‘Candidatus Phytoplasma asteris’ and Mungbean Yellow Mosaic India Virus Affecting Soya Bean Crop in India

Suspected phytoplasma and virus‐like symptoms of little leaf, yellow mosaic and witches’ broom were recorded on soya bean and two weed species (Digitaria sanguinalis and Parthenium hysterophorus), at experimental fields of Indian Agricultural Research Institute, New Delhi, India, in August–September 2013. The phytoplasma aetiology was confirmed in symptomatic soya bean and both the weed species by direct and nested PCR assays with phytoplasma‐specific universal primer pairs (P1/P6 and R16F2n/R16R2n). One major leafhopper species viz. Empoasca motti Pruthi feeding on symptomatic soya bean plants was also found phytoplasma positive in nested PCR assays. Sequencing BLASTn search analysis and phylogenetic analysis revealed that 16Sr DNA sequences of phytoplasma isolates of soya bean, weeds and leafhoppers had 99% sequence identity among themselves and were related to strains of ‘Candidatus Phytoplasma asteris’. PCR assays with Mungbean yellow mosaic India virus (MYMIV) coat‐protein‐specific primers yielded an amplicon of approximately 770 bp both from symptomatic soya bean and from whiteflies (Bemisia tabaci) feeding on soya bean, confirmed the presence of MYMIV in soya bean and whitefly. Hence, this study suggested the mixed infection of MYMIV and ‘Ca. P. asteris’ with soya bean yellow leaf and witches’ broom syndrome. The two weed species (D. sanguinalis and P. hysterophorus) were recorded as putative alternative hosts for ‘Ca. P. asteris’ soya bean Indian strain. However, the leafhopper E. motti was recorded as putative vector for the identified soya bean phytoplasma isolate, and the whitefly (B. tabaci) was identified as vector of MYMIV which belonged to Asia‐II‐1 genotype.     

Multilocus sequence typing confirms the close genetic interrelatedness of three distinct flavescence dorée phytoplasma strain clusters and group 16SrV phytoplasmas infecting grapevine and alder in Europe

Vineyards of southern France and northern Italy are affected by the flavescence dorée (FD) phytoplasma, a quarantine pathogen transmitted by the leafhopper of Nearctic origin Scaphoideus titanus. To better trace propagation of FD strains and identify possible passage between the vineyard and wild plant compartments, molecular typing of phytoplasma strains was applied. The sequences of the two genetic loci map and uvrB-degV, along with the sequence of the secY gene, were determined among a collection of FD and FD-related phytoplasmas infecting grapevine, alder, elm, blackberry, and Spanish broom in Europe. Sequence comparisons and phylogenetic analyses consistently indicated the existence of three FD phytoplasma strain clusters. Strain cluster FD1 (comprising isolate FD70) displayed low variability and represented 17% of the disease cases in the French vineyard, with a higher incidence of the cases in southwestern France. Strain cluster FD2 (comprising isolates FD92 and FD-D) displayed no variability and was detected both in France (83% of the cases) and in Italy, whereas the more-variable strain cluster FD3 (comprising isolate FD-C) was detected only in Italy. The clonal property of FD2 and its wide distribution are consistent with diffusion through propagation of infected-plant material. German Palatinate grapevine yellows phytoplasmas (PGY) appeared variable and were often related to some of the alder phytoplasmas (AldY) detected in Italy and France. Finally, phylogenetic analyses concluded that FD, PGY, and AldY were members of the same phylogenetic subclade, which may have originated in Europe.     

Flavescence dorée phytoplasma deregulates stomatal control of photosynthesis in Vitis vinifera

Flavescence dorée (FD) is among the major grapevine diseases causing high management costs; curative methods against FD are unavailable. In FD‐infected plants, decrease in photosynthesis is usually recorded, but deregulation in stomatal control of leaf gas exchange during FD infection and recovery is unknown. We measured the seasonal time course of gas exchange rates in two cultivars (‘Barbera’ and ‘Nebbiolo’) during the term of 1 year when grapevines experienced a water stress and another with no drought, with difference in gas exchange rates in response to FD infection and recovery as assessed by symptom observation and phytoplasma detection through PCR analysis. Chlorophyll fluorescence was also evaluated at the time of maximum symptom severity in ‘Barbera’, the cultivar showing the most severe stress response to FD infection, causing the highest damage in vineyards of north‐western Italy. In FD‐infected plants, net photosynthesis and transpiration gradually decreased during the season, more during the no drought year than during drought. During recovery, healthy (PCR negative) plants infected 2 years before, but not those infected an year before, regained the gas exchange performances to the level as measured before infection. The relationships between stomatal conductance and the residual leaf intercellular CO₂ concentration (c_i) discriminated healthy versus FD‐infected and recovered plants; at the same c_i, FD‐infected leaves had higher non‐photochemical quenching than healthy ones. We conclude that metabolic, not stomatal, leaf gas exchange limitation in FD‐infected and recovered grapevines is the basis of plant response to FD disease. In addition, we also suggest that such response is dependent upon water stress, by showing that water stress superimposes on FD infection in terms of stomatal and metabolic non‐stomatal limitations to carbon assimilation.     

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.     

Multilocus Sequence Typing Confirms the Close Genetic Interrelatedness of Three Distinct Flavescence Dorée Phytoplasma Strain Clusters and Group 16SrV Phytoplasmas Infecting Grapevine and Alder in Europe

Vineyards of southern France and northern Italy are affected by the flavescence dorée (FD) phytoplasma, a quarantine pathogen transmitted by the leafhopper of Nearctic origin Scaphoideus titanus. To better trace propagation of FD strains and identify possible passage between the vineyard and wild plant compartments, molecular typing of phytoplasma strains was applied. The sequences of the two genetic loci map and uvrB-degV, along with the sequence of the secY gene, were determined among a collection of FD and FD-related phytoplasmas infecting grapevine, alder, elm, blackberry, and Spanish broom in Europe. Sequence comparisons and phylogenetic analyses consistently indicated the existence of three FD phytoplasma strain clusters. Strain cluster FD1 (comprising isolate FD70) displayed low variability and represented 17% of the disease cases in the French vineyard, with a higher incidence of the cases in southwestern France. Strain cluster FD2 (comprising isolates FD92 and FD-D) displayed no variability and was detected both in France (83% of the cases) and in Italy, whereas the more-variable strain cluster FD3 (comprising isolate FD-C) was detected only in Italy. The clonal property of FD2 and its wide distribution are consistent with diffusion through propagation of infected-plant material. German Palatinate grapevine yellows phytoplasmas (PGY) appeared variable and were often related to some of the alder phytoplasmas (AldY) detected in Italy and France. Finally, phylogenetic analyses concluded that FD, PGY, and AldY were members of the same phylogenetic subclade, which may have originated in Europe.     

New insights in phytoplasma‐vector interaction: acquisition and inoculation of flavescence dorée phytoplasma by Scaphoideus titanus adults in a short window of time

The leafhopper Scaphoideus titanus is able to transmit 16SrV phytoplasmas agents of grapevine's flavescence dorée (FD) within 30–45 days, following an acquisition access period (AAP) of a few days feeding on infected plants as a nymph, a latency period (LP) of 3–5 weeks becoming meanwhile an adult, and an inoculation access period (IAP) of a few days on healthy plants. However, several aspects of FD epidemiology suggest how the whole transmission process may take less time, and may start directly with adults of the insect vector. Transmission experiments have been set up under lab condition. Phytoplasma‐free S. titanus adults were placed on broad bean (BB) plants (Vicia faba) infected by FD‐C (16SrV‐C) phytoplasmas for an AAP = 7 days. Afterwards, they were immediately moved onto healthy BB for IAP, which were changed every 7 days, obtaining three timings of inoculation: IAP 1, IAP 2 and IAP 3, lasting 7, 14 and 21 days from the end of AAP, respectively. DNA was extracted from plants and insects, and PCR tests were performed to identify FD phytoplasmas. Insects were dissected and fluorescence in situ hybridisation was made to detect the presence of phytoplasmas in midguts and salivary glands. The rate of infection in insects ranged 46–68% without significant differences among IAPs. Inoculation in plants succeeded in all IAPs, at a rate of 16–23% (no significant differences). Phytoplasma load was significantly higher in IAP 3 than IAP 1–2 for both plants and insects. Phytoplasmas were identified both in midgut and salivary glands of S. titanus at all IAP times. The possible implications of these results in the epidemiology of flavescence dorée are discussed.     

Dispersal patterns and chromatic response of Scaphoideus titanus Ball (Homoptera Cicadellidae), vector of the phytoplasma agent of grapevine flavescence dorée

Abstract 1 Scaphoideus titanus Ball, a nearctic leafhopper introduced into Europe in the 1950s, is known to be the vector of the phytoplasma agent of flavescence dorée (FD), a persistent disease of grapevine. Knowledge of its dispersal patterns is thus very important to prevent disease outbreaks. 2 Yellow sticky traps were used to study the seasonal flight activity of S. titanus, its vertical flight, its movement outside the vineyard and the influence of plant density. Sticky traps of different colours (yellow, red, blue, and white) were also compared. The behaviour of males and females was tested for all those conditions. 3 Abundance was greater in normal than in low plant density conditions, and a positive relationship was found between number of plants per square metre and presence of S. titanus. Leafhoppers did not appear capable of spreading significantly outside a vineyard. Few individuals were trapped above the canopy. Red sticky traps caught more individuals than white, yellow or blue, with the latter showing a poor attractiveness. Sex ratio was almost always male biased. 4 Scaphoideus titanus is monophagous and appears incapable of great dispersal away from its host plant, and females are less likely to fly than males. Further studies on the influence of different factors on the behaviour of this leafhopper are suggested.     

Occurrence,Molecular Characterization and Vector Transmission of a Phytoplasma Associated with Rapeseed Phyllody in Iran

Symptoms of rapeseed phyllody were observed in rapeseed fields of Fars, Ghazvin, Isfahan, Kerman and Yazd provinces in Iran. Circulifer haematoceps leafhoppers testing positive for phytoplasma in polymerase chain reaction (PCR) successfully transmitted a rapeseed phyllody phytoplasma isolate from Zarghan (Fars province) to healthy rapeseed plants directly after collection in the field or after acquisition feeding on infected rapeseed in the greenhouse. The disease agent was transmitted by the same leafhopper from rape to periwinkle, sesame, stock, mustard, radish and rocket plants causing phytoplasma‐type symptoms in these plants. PCR assays using phytoplasma‐specific primer pair P1/P7 or nested PCR using primers P1/P7 followed by R16F2n/R2, amplified products of expected size (1.8 and 1.2 kbp, respectively) from symptomatic rapeseed plants and C. haematoceps specimens. Restriction fragment length polymorphism analysis of amplification products of nested PCR and putative restriction site analysis of 16S rRNA gene indicated the presence of aster yellows‐related phytoplasmas (16SrI‐B) in naturally and experimentally infected rapeseed plants and in samples of C. haematoceps collected in affected rapeseed fields. Sequence homology and phylogenetic analysis of 16S rRNA gene confirmed that the associated phytoplasma detected in Zarghan rapeseed plant is closer to the members of the subgroup 16SrI‐B than to other members of the AY group. This is the first report of natural occurrence and characterization of rapeseed phyllody phytoplasma, including its vector identification, in Iran.     

Euscelis incisus (Cicadellidae,Deltocephalinae), a natural vector of 16SrIII‐B phytoplasma causing multiple inflorescence disease of Cirsium arvense

We investigated multiple inflorescence disease of Cirsium arvense (CMI) and its association with phytoplasmas of the 16SrIII‐B subgroup, potential natural vector(s) and reservoir plant(s). From five locations in northern Serbia, 27 plants of C. arvense, 1 C. vulgare and 3 Carduus acanthoides with symptoms of multiple inflorescences (MIs) were collected and tested for 16SrIII group phytoplasmas. All symptomatic plants were found to be infected. Tentative reservoir plants and insect vectors were collected at a Dobanovci site where the continuous presence of CMI disease was recorded. Among the 19 most abundant plant species submitted to phytoplasma testing, all symptomless, the presence of the 16SrIII group was detected only in two legumes: Lathyrus tuberosus (2/5) and L. aphaca (1/5). Among 19 insect species from six families of Auchenorrhyncha, the deltocephalid leafhopper Euscelis incisus was the only insect carrying a 16SrIII phytoplasma (10% of analysed individuals). Transmission trials were performed with naturally infected E. incisus adults of the summer generation and with a laboratory population reared on red clover. After an acquisition period of 48 h on C. arvense symptomatic for MIs and a latent period of 28 days, 83% of the E. incisus adults (300/360) were infected with CMI phytoplasma. In two transmission tests, the leafhoppers successfully transmitted the phytoplasma to exposed plants (C. arvense and periwinkle), proving its role as a natural vector. Test plants of C. arvense infected with the 16SrIII‐B phytoplasma expressed typical symptoms similar to those observed in the field, such as MIs or the absence of flowering, shortened internodes and plant desiccation. Typical symptoms in infected periwinkles were virescence and phyllody. The molecular characterisation of the CMI phytoplasma isolates from diseased and asymptomatic field‐collected plants, vectors, and test plants was performed by sequence analyses of the 16S rRNA, rpl22‐rps3 and rpl15‐secY genes. Phylogenetic analyses of other members of the 16SrIII group of phytoplasmas indicated closest relatedness with clover yellow edge phytoplasma (CYE) of the 16SrIII‐B subgroup.     

Irradiation of Maconellicoccus hirsutus (Homoptera: Pseudococcidae) for phytosanitation of agricultural commodities

Studies on the tolerance of pink hibiscus mealybug, Maconellicoccus hirsutus (Green), to ionizing irradiation were undertaken to determine the dose needed to disinfest commodities of this pest. Overall, radiotolerance of M. hirsutus was found to increase with maturity. Target doses of 50 Gy reduced eclosion of eggs to <50%, but doses as great as 750 Gy did not eliminate hatching during the study. At 100 Gy, M. hirsutus eggs, crawlers, and nymphs were controlled, because progeny were not produced despite crawlers and nymphs living for much longer periods than unexposed individuals. Fecundity of treated crawlers and nymphs was greatly impacted by treatment of 100 Gy; crawlers developing into adults produced no eggs, and 10 adults of 3,983 treated nymphs (0.25%) produced 309 eggs. Few adult females exposed as nymphs deposited eggs because male nymphs died during development, which left the females unfertilized. By comparison, 89% of female nymphs treated at 100 Gy and mated as adults with nonirradiated males produced a total of 1,447 eggs (19 eggs per female). Evidence from this study suggests M. hirsutus reproduces sexually, not parthenogenetically. Adults, the most resistant stage, exposed to target doses of 100 Gy produced eggs that were 1.2% viable, from which a small portion of individuals successfully completed development and produced progeny. A target dose of 250 Gy was sufficient to control adult M. hirsutus because, at that dose, none of the eggs produced by 3,093 irradiated adults eclosed. The minimum dose needed to ensure quarantine security is between 100 and 250 Gy.     

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.     

Study of transovarial passage of the oat sterile-dwarf virus

The possibility of passage of the oat sterile-dwarf virus by the leafhopper Calligypona pellucida F. as vector was studied in the years 1961–1964. Females infected in a greenhouse as well as in nature produced 6,000 eggs from which some 3,500 nymphs hatched. Of these, 3,423 were subjected to individual testing on oat seedlings. In seven cases (0,2%) a transfer of the studied virus through eggs of infected females to their offspring was observed. The infectivity began to manifest itself 9–23 days after hatching.