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A phytoplasma strain associated with pot marigold phyllody disease in Fars province of Iran was identified and characterized by molecular analyses and biological studies. Nested polymerase chain reaction using universal P1/P7 primer pair followed by R16F2n/R2, confirmed the presence of phytoplasmas in the symptomatic plants only, and in Psammotettix striatus leafhopper. BLAST search, phylogenetic analysis, and virtual RFLP patterns of cloned amplicons allowed to classify the pot marigold phyllody and P. striatus leafhopper phytoplasmas in the 16SrI-S subgroup. The phytoplasma was transmitted from infected pot marigold to periwinkle plants by dodder inducing symptoms typical of phytoplasma presence. P. striatus transmitted this phyllody phytoplasma to healthy pot marigold and periwinkle plants after its collection in the infected fields or after acquisition feeding on infected pot marigold plants under insect-proof greenhouse conditions. This is the first report of 16SrI-S phytoplasma associated with phyllody of pot marigold and of a phytoplasma of this ribosomal subgroup in Iran.

Quantitative estimates of vector populations and their infectivity in the wild and in cultivated compartments of agroecosystems have been carried out to elucidate the role of the wild compartment in the epidemiology of Flavescence dorée (FD). Seven sites were selected for the investigations in the Piedmont Region of Italy. They were characterized by a high variety of agricultural and ecological landscape features, and included a vineyard surrounded by wild vegetation. In order to describe abundance and prevalence of FD-infected vectors in the cultivated and wild compartments of the vineyard agroecosystem, adults of Scaphoideus titanus were collected by yellow sticky traps inside and outside the vineyard over the period July 10th–September 9th, 2015. They were counted and singly analyzed for the presence of FD phytoplasmas by PCR. Multifactorial correlations among vector population level, prevalence of infected insects inside and outside the vineyards, disease prevalence in cultivated and wild Vitis plants, and location of wild Vitis plants with respect to the vineyard were analyzed. Abundance of S. titanus adults significantly decreased from the end of July onwards, particularly inside the vineyard (average range 22.7 ± 2.5 insects/trap). Percentage of FD-positive S. titanus was significantly higher outside the vineyard (up to 48% on average) compared to inside the vineyard (up to 34% on average), and increased during the season in both compartments.

Objectives Spiroplasma citri is a bacterium with a wide host range and is the causal agent of citrus stubborn and brittle root diseases of citrus and horseradish, respectively. S. citri is transmitted in a circulative, persistent manner by the beet leafhopper, Neoaliturus (Circulifer) tenellus (Baker), in North America. Five strains of S. citri were cultured from citrus, horseradish, and N. tenellus from different habitats and times. DNA from cultures were sequenced and genome assembled to expand the database to improve detection assays and better understand its genetics and evolution. Data description The whole genome sequence of five strains of S. citri are described herein. The S. citri chromosome was circularized for all five strains and ranged from 1,576,550 to 1,742,208 bp with a G + C content of 25.4–25.6%. Characterization of extrachromosomal DNAs resulted in identification of one or two plasmids, with a G + C content of 23.3 to 27.6%, from plant hosts; and eight or nine plasmids, with a G + C content of 21.65 to 29.19%, from N. tenellus . Total genome size ranged from 1,611,714 to 1,832,173 bp from plants and 1,968,976 to 2,155,613 bp from the leafhopper. All sequence data has been deposited in DDBJ/ENA/GenBank under the accession numbers CP046368-CP046373 and CP047426-CP047446.

The dispersion of Scaphoideus titanus Ball adults from wild to cultivated grapevines was studied using a novel mark–capture technique. The crowns of wild grapevines located at a distance from vineyards ranging from 5 to 330 m were sprayed with a water solution of either cow milk (marker: casein) or chicken egg whites (marker: albumin) and insects captured in yellow sticky traps placed on the canopy of grapes were analyzed via an indirect ELISA for markers’ identification. Data were subject to exponential regression as a function of distance from wild grapevine, and to spatial interpolation (Inverse Distance Weighted and Kernel interpolation with barriers) using ArcGIS Desktop 10.1 software. The influence of rainfall and time elapsed after marking on markers’ effectiveness, and the different dispersion of males and females were studied with regression analyses. Of a total of 5417 insects analyzed, 43% were positive to egg; whereas 18% of 536 tested resulted marked with milk. No influence of rainfall or time elapsed was observed for egg, whereas milk was affected by time. Males and females showed no difference in dispersal. Marked adults decreased exponentially along with distance from wild grapevine and up to 80% of them were captured within 30 m. However, there was evidence of long-range dispersal up to 330 m. The interpolation maps showed a clear clustering of marked S. titanus close to the treated wild grapevine, and the pathways to the vineyards did not always seem to go along straight lines but mainly along ecological corridors. S. titanus adults are therefore capable of dispersing from wild to cultivated grapevine, and this may affect pest management strategies.

The knockdown and toxic effects of insecticides of different chemical groups and modes of action registered for citrus in Brazil were investigated for effective control of Bucephalogonia xanthophis, a sharpshooter vector of Xylella fastidiosa in citrus. The active ingredients dimethoate (1.2 mL/1.2L), imidacloprid (0.24 mL/1.2L) and lambda-cyhalothrin (0.24 mL/1.2L), as well as a control (water), were sprayed onto branches of potted citrus nursery trees to evaluate the effect of residual contact. The insects were confined on sprayed branches by using sleeve cages, in groups of 10 per branch (5 branches/treatment). Lambdacyhalothrin showed a knockdown effect on B. xanthophis (>70% mortality within 2 h of exposure), and the residues were effective for approximately one wk. Imidacloprid, lambdacyhalothrin and dimethoate suppressed the vector populations for up to 3 wk after application, when the insects were exposed to sprayed plants for at least 24 h. In another experiment, 2 neonicotinoid insecticides (thiamethoxam and imidacloprid) were applied by soil drench to potted nursery trees, in order to study their systemic effect, i.e., mortality by ingestion on sharpshooter adults. Thiamethoxam and imidacloprid effectively controlled the vectors at all concentrations tested, when the insects were exposed to treated plants for 24 h (>80% mortality) or 48 h (near 100% mortality). The knockdown effect of thiamethoxam and lambda-cyhalothrin might be particularly important to prevent vector transmission of X. fastidiosa in citrus groves.

The differences between the seasonal occurrence and likelihood of being infected by FD phytoplasmas, of male and female Scaphoideus titanus Ball, were investigated. Sex ratio (male: female) was calculated by counting males and females sampled by means of yellow sticky traps and sweep-nets and from adults derived from hatched eggs in field-collected grapevine wood. PCR essays were performed to test differences in infection between genders. Overall, the sex ratio on sticky traps was significantly more male biased (1.99:1) if compared to net sweeping (0.62:1) and laboratory rearing (0.60:1). The peak of male presence was recorded in the middle of July in laboratory rearing and sweep net, and in the middle of August on sticky traps; the maximum presence of females was detected at the end of July in laboratory rearing, and at the end of August in sweep net samplings and on sticky traps. The seasonal sex ratio was more male biased at the beginning in laboratory rearing (1.50:1) and sticky traps (9:1), and then decreased in favor of females at the end of the sampling period, both in laboratory rearing (0.17:1) and in sticky traps (0.07:1). This trend was significantly less skewed, although similar, in sweep net samplings that recorded a sex ratio of 1:1 and 0.16:1 at the beginning and at the end of the sampling period, respectively. Concerning phytoplasma detection, an interaction between gender and sampling period was observed, the males showing a peak of infected individuals later in the season (35%). Some possible behavioral explanations of the data obtained are given.

Ultrastructural studies using scanning electron microscopy (SEM), negative-staining transmission electron microscopy (TEM), and thin-sectioning TEM on four species of Spiroplasma, in vitro and/or in vivo, indicated that their helices commonly possess one tapered end (tip structure) and one blunt or round end. These tip structures appeared morphologically different from the rest of the helix, exhibiting an electron-dense conical or rod-shaped core. In thin sections of the midgut of the leafhopper Dalbulus elimatus, the tip structures of Spiroplasma kunkelii in the midgut lumen were mostly aligned between microvilli, perpendicular to the apical plasma membrane of epithelial cells. These tip structures appeared frequently attached or closely apposed to the plasma membrane, in which cup-shaped invaginations close to the tips were observed. Pleomorphic forms of spiroplasma, enclosed in membranous vesicles, were found in the cytoplasm of the midgut epithelial cells. These findings suggest that the tip structure may be involved in the orientation and attachment of spiroplasma helices in relation to their host cells, and thus may be functionally comparable to the \"attachment organelle\" of mycoplasmas. Additionally, pili-like structures were observed by negative-staining TEM on the surface of Spiroplasma melliferum, and in thin sections of S. kunkelii infecting the leafhopper vector Dalbulus gelbus.

A disease with symptoms similar to elm yellows (EY) was noticed in the early 1990s in suburban Chicago, IL. More than 1,000 mature American elms (Ulmus americana) have since died. Infected trees varied in the incidence and severity of canopy yellowing, leaf epinasty, butterscotch discoloration, and wintergreen odor of the phloem, but all developed a sparse and clumpy crown, uniformly necrotic phloem, and died within 2 years of showing canopy symptoms. Because symptoms were expressed irregularly and phytoplasma detection results by a commercial diagnostic company were inconsistent, a study was initiated to determine if EY phytoplasma was the causal agent. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods using universal or EY phytoplasma specific primers were employed to detect putative phytoplasma(s) associated with 10 trees of varied disease severity within the outbreak region and 10 asymptomatic trees from an uninfected area (controls). Nested PCR using universal primers revealed that 90% of trees from the outbreak region were positive for phytoplasma while asymptomatic elms from another location (controls) tested negative. Phytoplasma-positive trees ranged in disease severity from 1 (asymptomatic) to 5 (near death). Inner bark samples chiseled from the lower trunk had higher phytoplasma detection rates than foliage or drill shavings. RFLP analyses and DNA sequencing of 16S rDNA indicated that the phytoplasma recovered from dying elms in Arlington Heights is not related to the reference EY phytoplasma (group16SrV). It is most closely related to clover proliferation (CP) phy-toplasma (group 16SrVI), and we have designated it Illinois Elm Yellows (ILEY) phytoplasma, and assigned it to a new taxonomic subgroup (16SrVI-C). EY phytoplasma was not detected in any samples, but two ILEY phytoplasma positive trees also were positive for aster yellows (AY) phytoplasma. ILEY phytoplasma was not detected in local leafhopper populations trapped in elm trees between May and September 2000. This is the first report of a phytoplasma related to CP phytoplasma causing elm yellows disease symptoms.

The liquid excretion and survival of the sharpshooters Dilobopterus costalimai Young and Oncometopia facialis (Signoret), vectors of Xylella fastidiosa in citrus, were measured on various host plants as an indirect approach to assess their feeding and performance on these hosts and determine suitable plants for laboratory rearing. Adult females of D. costalimai showed the highest excretion rate on Vernonia condensata (Asteraceae). O. facialisexcreted larger volumes on three species of Vernonia and on Lantana camara (Verbenaceae). On average, single D. costalimai females excreted a liquid volume equivalent to 292 times its body volume per day when feeding on V. condensata, whereas O. facialis females excreted 430 times their body volume on the same host. In contrast, the excretion rates of D. costalimai and O. facialis females on Citrus sinensis did not exceed 248 and 140 times their body volume per day, respectively. The mortality of adults after 96 h was lower on hosts upon which higher liquid volumes were excreted; therefore, there is a positive relationship between the excretion rate by the sharpshooters and their nutritional adequacy to hosts. V. condensata is a suitable host to maintain adult populations of both sharpshooters in the laboratory.

DNA extraction and storage methods have been evaluated with laboratory-reared leafhoppers and/or field-collected leafhoppers and psyllids. Detection of four different phytopathogenic phytoplasmas, belonging to three taxonomic groups, has been achieved by several direct or nested polymerase chain reaction (PCR) methods with such DNA extracts. Reactions differed in both the 16/23S ribosomal primer pairs used and the specific assay and cycling conditions. Merits and possible hindrances of the various primer pairs, in relation to insect DNA extracts, are discussed. However, identification of the phytoplasma(s) necessarily relied on comparison of the polymorphism in length of the amplified DNA fragments obtained by restriction with appropriate endonucleases. Endonuclease digestion is crucial for determining the identity (subgroup affiliation) of phytoplasmas of the same groups that can be carried by an individual vector.