Cristina Marzachì

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 (plant or insect) 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.

Striking Diversity of vmp1, a Variable Gene Encoding a Putative Membrane Protein of the Stolbur Phytoplasma

ABSTRACT Studies of phytoplasma-insect vector interactions and epidemiological surveys of plant yellows associated with the stolbur phytoplasma (StolP) require the identification of relevant candidate genes and typing markers. A recent StolP genome survey identified a partial coding sequence, SR01H10, having no homologue in the “Candidatus Phytoplasma asteris” genome but sharing low similarity with a variable surface protein of animal mycoplasmas. The complete coding sequence and its genetic environment have been fully characterized by chromosome walking. The vmp1 gene encodes a protein of 557 amino acids predicted to possess a putative signal peptide and a potential C-terminal transmembrane domain. The mature 57.8-kDa VMP1 protein is likely to be anchored in the phytoplasma membrane with a large N-terminal hydrophilic part exposed to the phytoplasma cell surface. Southern blotting experiments detected multiple sequences homologous to vmp1 in the genomes of nine StolP isolates. vmp1 is variable in size, and eight different vmp1 RsaI restriction fragment length polymorphism types could be distinguished among 12 StolP isolates. Comparison of vmp1 sequences revealed that insertions in largest forms of the gene encode an additional copy of a repeated domain of 81 amino acids, while variations in 11-bp repeats led to gene disruption in two StolP isolates. vmp1 appeared to be much more variable than three housekeeping genes involved in protein translation, maturation, and secretion and may therefore be involved in phytoplasma-host interactions.

The Major Antigenic Membrane Protein of “Candidatus Phytoplasma asteris” Selectively Interacts with ATP Synthase and Actin of Leafhopper Vectors

Phytoplasmas, uncultivable phloem-limited phytopathogenic wall-less bacteria, represent a major threat to agriculture worldwide. They are transmitted in a persistent, propagative manner by phloem-sucking Hemipteran insects. Phytoplasma membrane proteins are in direct contact with hosts and are presumably involved in determining vector specificity. Such a role has been proposed for phytoplasma transmembrane proteins encoded by circular extrachromosomal elements, at least one of which is a plasmid. Little is known about the interactions between major phytoplasma antigenic membrane protein (Amp) and insect vector proteins. The aims of our work were to identify vector proteins interacting with Amp and to investigate their role in transmission specificity. In controlled transmission experiments, four Hemipteran species were identified as vectors of “Candidatus Phytoplasma asteris”, the chrysanthemum yellows phytoplasmas (CYP) strain, and three others as non-vectors. Interactions between a labelled (recombinant) CYP Amp and insect proteins were analysed by far Western blots and affinity chromatography. Amp interacted specifically with a few proteins from vector species only. Among Amp-binding vector proteins, actin and both the α and β subunits of ATP synthase were identified by mass spectrometry and Western blots. Immunofluorescence confocal microscopy and Western blots of plasma membrane and mitochondrial fractions confirmed the localisation of ATP synthase, generally known as a mitochondrial protein, in plasma membranes of midgut and salivary gland cells in the vector Euscelidius variegatus. The vector-specific interaction between phytoplasma Amp and insect ATP synthase is demonstrated for the first time, and this work also supports the hypothesis that host actin is involved in the internalization and intracellular motility of phytoplasmas within their vectors. Phytoplasma Amp is hypothesized to play a crucial role in insect transmission specificity.

Interrelationships between "Candidatus Phytoplasma asteris" and its leafhopper vectors (Homoptera: Cicadellidae).

Abstract 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.

Characterization of Bois Noir Isolates by Restriction Fragment Length Polymorphism of a Stolbur-Specific Putative Membrane Protein Gene

Bois noir phytoplasma (BNp), widespread in wine-producing areas of Europe and endemic in France and Italy, is classified in the 16SrXII-A subgroup, whose members are referred to as Stolbur phytoplasmas. The 16S rDNA gene of Stolbur phytoplasma shows low variability, and few non-ribosomal genes are available as markers to assess variation among isolates. We used the Stolbur-specific stol-1H10 gene, encoding a putative membrane-exposed protein, to investigate genetic diversity of French and Italian BNp isolates from plants and insects. Amplification of stol-1H10 from infected grapevines, weeds, and Hyalesthes obsoletus produced fragments of three sizes, and restriction fragment length polymorphism analysis divided these amplicons further into 12 profiles (V1 to V12). French BNp isolates were more variable than Italian ones, and different profiles were present in infected grapevines from France and Italy. Isolate V3, most abundant among Italian affected grapes but present among French ones, was found in one Urtica dioica sample and in all H. obsoletus collected on this species. Four Italian-specific profiles were represented among infected Convolvulus arvensis, the most frequent of which (V12) was also detected in H. obsoletus collected on this species. Most of the variability in the stol-1H10 sequence was associated with type II on the tuf gene.

A multidisciplinary study on the effects of phloem-limited viruses on the agronomical performance and berry quality of Vitis vinifera cv. Nebbiolo

Viral infections are known to have a detrimental effect on grapevine yield and performance, but there is still a lack of knowledge about their effect on the quality and safety of end products. Vines of Vitis vinifera cv. Nebbiolo clone 308, affected simultaneously by Grapevine leafroll-associated virus 1 (GLRaV-1), Grapevine virus A (GVA), and Rupestris stem pitting associated virus (RSPaV), were subjected to integrated analyses of agronomical performance, grape berry characteristics, instrumental texture profile, and proteome profiling. The comparison of performance and grape quality of healthy and infected vines cultivated in a commercial vineyard revealed similar shoot fertility, number of clusters, total yield, with significant differences in titratable acidity, and resveratrol content. Also some texture parameters such as cohesiveness and resilience were altered in berries of infected plants. The proteomic analysis of skin and pulp visualized about 400 spots. The ANOVA analysis on 2D gels revealed significant differences among healthy and virus-infected grape berries for 12 pulp spots and 7 skin spots. Virus infection mainly influenced proteins involved in the response to oxidative stress in the berry skin, and proteins involved in cell structure metabolism in the pulp.

DNA-based methods for the detection and the identification of phytoplasmas in insect vector extracts.

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.

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

ABSTRACT 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 Vicia faba (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 a low 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.

Detection of Flavescence Dorée Phytoplasma in Grapevine by Reverse-Transcription PCR

Flavescence dorée (FD) is the most serious phytoplasma disease of grapevine. This report describes a novel method of detecting FD phytoplasma based on reverse-transcription polymerase chain reaction (RT-PCR) on 16S ribosomal RNA (16SrRNA) which will greatly improve mass screening of infected grapevines. A rapid protocol for extracting sap from whole leaves or midveins and successive one-tube amplification by RT-PCR was applied to grapevine samples with or without symptoms collected from different areas of Piedmont (northwestern Italy). Results were compared with those obtained using one of the current diagnostic methods that utilizes nested PCR on phytoplasma DNA-enriched preparations. A Cohens kappa index of 0.76 indicated a substantial agreement between the two sets of results. The RT-PCR method has the advantage of being a rapid, reliable, and sensitive assay for large-scale screening of grapevines.

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

Phytoplasmas 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.