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

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

Transmission of Grapevine virus A and Grapevine leafroll-associated virus 1 and 3 by Heliococcus bohemicus (Hemiptera: Pseudococcidae) Nymphs From Plants With Mixed Infections.

Mealybugs (Hemiptera: Pseudococcidae) represent a serious threat for viticulture as vectors of phloem-restricted viruses associated with the grapevine rugose wood and leafroll diseases. Heliococcus bohemicus (Sulc) is known to be involved in the spread of these two viral diseases, being a vector of the Grapevine virus A (GVA) and the Grapevine leafroll-associated virus 1 and 3 (GLRaV-1 and GLRaV-3). This study investigated the acquisition and transmission efficiency of H. bohemicus fed on mixed-infected plants. Nymphs were field-collected onto GVA, GLRaV-1, and GLRaV-3 multiple-infected grapevines in two vineyards in North-Western Italy, and were used in transmission experiments under controlled conditions. Even if most of the collected nymphs were positive to at least one virus, transmission occurred only to a low number of test grapevines. The transmission frequency of GLRaV-3 was the highest, whereas GVA was transmitted to few test plants. The transmission of multiple viruses occurred at low rates, and nymphs that acquired all the three viruses then failed to transmit them together. Statistical analyses showed that the three viruses were independently acquired and transmitted by H. bohemicus and neither synergistic nor antagonistic interactions occurred among them. GVA and GLRaVs transmission efficiencies by H. bohemicus were lower than those reported for other mealybug vectors. This finding is consistent with the slow spread of leafroll and rugose wood diseases observed in Northern Italy, where H. bohemicus is the predominant vector species.

Decreasing global transcript levels over time suggest that phytoplasma cells enter stationary phase during plant and insect colonization.

To highlight different transcriptional behaviors of the phytoplasma in the plant and animal host, expression of 14 genes of "Candidatus Phytoplasma asteris," chrysanthemum yellows strain, was investigated at different times following the infection of a plant host (Arabidopsis thaliana) and two insect vector species (Macrosteles quadripunctulatus and Euscelidius variegatus). Target genes were selected among those encoding antigenic membrane proteins, membrane transporters, secreted proteins, and general enzymes. Transcripts were detected for all analyzed genes in the three hosts; in particular, those encoding the antigenic membrane protein Amp, elements of the mechanosensitive channel, and two of the four secreted proteins (SAP54 and TENGU) were highly accumulated, suggesting that they play important roles in phytoplasma physiology during the infection cycle. Most transcripts were present at higher abundance in the plant host than in the insect hosts. Generally, transcript levels of the selected genes decreased significantly during infection of A. thaliana and M. quadripunctulatus but were more constant in E. variegatus. Such decreases may be explained by the fact that only a fraction of the phytoplasma population was transcribing, while the remaining part was aging to a stationary phase. This strategy might improve long-term survival, thereby increasing the likelihood that the pathogen may be acquired by a vector and/or inoculated to a healthy plant.

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.

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.

Interrelationships between "Candidatus 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.

Epidemiology of flavescence dorée in vineyards in northwestern Italy.

ABSTRACT A serious outbreak of flavescence dorée (FD) was reported in Piemonte, northwestern Italy, in 1998, and since then, the disease has compromised the economy of this traditional wine-growing area, even following the application of compulsory insecticide treatments to control Scaphoideus titanus, the vector of the causal phytoplasma. Affected vines show severe symptoms, varying according to the cultivar, and are rogued to reduce disease spread. Following winter and pruning, a previously affected vine may appear symptomless and free of phytoplasmas in its aerial as well as its root system, even by nested-polymerase chain reaction assays. Such plants are considered to be "recovered". Since 1998 homogenous data on the incidence of newly infected, healthy, or recovered plants productivity, presence of vectors, and treatment schedules have been collected in seven severely affected vineyards of southern Piemonte for 5 years (1999 to 2003). Infectivity and recovery rates were also calculated each year. From 1999 to 2003, the average number of healthy plants decreased and the numbers of recovered plants and those with symptoms increased. Productivity of recovered vines, although lower than that of healthy ones, was always higher than that of vines with symptoms and was not influenced by the time elapsed from date of recovery. The relationships between the ln-transformed number of vectors trapped in the vineyards the previous year and the infection and the recovery rates were fitted by an exponential (R(2) = 0.95) and an asymptotic (R(2) = 0.93) model, respectively.

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 Cohen's 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.

Genetic variability among flavescence dorée phytoplasmas from different origins in Italy and France.

Flavescence dorée is a devastating disease of grapevine widespread in several countries in EU such as France, Italy and Spain. Genetic variability among 17 Italian and 3 French FD strains was investigated by RFLP analyses based on a fragment of the ribosomal protein operon and on the non-ribosomal DNA fragment FD9. RFLP analysis of the PCR amplified ribosomal protein fragment, coding for the 3' end of rpl22 and the entire rps3 genes, differentiated 4 rp-subgroups among the FD strains and 4 subgroups among the reference strains belonging to elm yellows group (16SrV). Sequencing and phylogenetic analysis of the same ribosomal protein DNA fragment validated the delineation of 4 distinct FD strain types derived by RFLP analyses. The results supported the differentiation based on analysis of the non-ribosomal DNA fragment FD9. The phylogenetic analysis further revealed relationships and a probable evolutionary trend among the FD strains and the other representatives of elm yellows group. All the FD strains together with the reference strains ALY, RuS and JWB formed a cluster very well distinct from the EY/ULW cluster. Moreover, ALY was shown to be more closely related to three FD strain types: the Lombardia/Piemonte, the French FD70, and the French FD88/Italian FD-D strain clusters.

Generation of RAPD-PCR primers for the identification of isolates of Glomus mosseae, an arbuscular mycorrhizal fungus.

Mycorrhizal fungi are usually identified on the basis of the morphological characters shown by fruit bodies, spores, vegetative mycelia or symbiotic structures. The development of molecular techniques provides a valuable and alternative approach to identify mycorrhizal fungi, especially when it is difficult to gather a sufficient number of data on morphological features. Short arbitrary oligonucleotides were used as primers for the amplification of genomic DNA extracted from spores of arbuscular fungi. The RAPD fingerprints showed banding patterns which allowed us to distinguish between species and even isolates within Glomales. In order to identify mycorrhizal fungi during their symbiotic phase, a nonpolymorphic RAPD band identified as marker for some isolates of Glomus mosseae was purified from agarose gels and cloned in a bluescript vector. The fragment was sequenced and specific primers (PO-M3) were designed for the mycorrhizal fungus. They specifically and successfully amplified the DNA not only from G. mosseae spores, but also from roots of pea, clover, leek and onion plants when they were colonized by G. mosseae isolates.

DNA probes for identification of the ectomycorrhizal fungus Tuber magnatum Pico.

The random amplified polymorphic DNA (RAPD) technique was used to develop DNA probes for the identification of ectomycorrhizal fungi belonging to the genus Tuber. RAPD fingerprinting revealed a high degree of interspecific variability and a low degree of intraspecific variability. One band (approximately 1.5 kb), consistently appearing when genomic DNA was amplified with an aspecific primer (OPA-18), was found to be a good marker for Tuber magnatum, and was used as a probe in Southern hybridization experiments. The specificity of the results suggests that this probe may be useful in developing specific primers for PCR amplifications.

Cloning of the maize rough dwarf virus genome: molecular confirmation of the plant-reovirus classification scheme and identification of two large nonoverlapping coding domains within a single genomic segment.

The segmented double-stranded RNA genome of maize rough dwarf virus, a plant-infecting reovirus of the genus Fijivirus, was cloned and partially characterized. Nucleotide sequence analysis of full-length cDNA clones corresponding to genomic segments S6, S7, and S8 revealed each segment to contain the conserved terminal oligonucleotide sequences (+) 5' AAGUUUUUU------UGUC 3' and adjacent, segment-specific, regions of inverted complementarity (inverted repeats), a structural motif previously reported for members of the genus Phytoreovirus. Genomic segment S6 was completely sequenced and found to consist of 2193 base pairs. Computer analysis indicated that the coding strand contained two large nonoverlapping open reading frames consisting of 363 and 310 codons and located in the 5'- and 3'-terminal domains, respectively. This was confirmed by cell-free translation studies with synthetic transcripts and denatured genomic RNA. However, only the product of the 5'-proximal open reading frame, a 40-kDa polypeptide, was efficiently expressed in vitro from the full-length S6 coding strand. This represents the first case in which a reovirus genomic segment was found to contain two large open reading frames in a nonoverlapping configuration, suggesting possible alternative strategies for regulation of gene expression by members of this genus. The combined results provide a molecular confirmation of the current classification scheme for plant-infecting reoviruses. Furthermore, the fact that the same terminal structural motif is conserved across genera provides additional evidence that these elements serve an important functional role during genome transcription or replication.

In vitro transcription of the double-stranded RNA genome of maize rough dwarf virus (Reoviridae).

An RNA-dependent RNA polymerase associated with particles of maize rough dwarf virus, a Fijivirus, was characterized using two in vitro assays differing in their energy regeneration systems. Optimum reaction rates occurred at pH 8.0 to 8.5 at 20 degrees C. The presence of virus and Mn2+ or Mg2+ was essential for enzyme activity; Mn2+ stimulated more incorporation events than Mg2+, at optimum concentrations of 2 to 4 mM and 4 mM, respectively. Incorporation was not affected by alpha-amanitin, actinomycin D or rifampicin. The products synthesized in vitro were single-stranded RNAs which hybridized specifically with the double-stranded genomic RNAs of five other reoviruses. The in vitro transcripts were also used to detect maize rough dwarf virus RNA in plants and in vector insects.

In vitro synthesis of double-stranded RNA by carnation cryptic virus-associated RNA-dependent RNA polymerase.

Partially purified carnation cryptic virus (CarCV) preparations possessed RNA-dependent RNA polymerase activity which was absent in comparable preparations from virus-free carnations. Enzyme activity was dependent upon the presence of virus particles, Mg2+, and the four ribonucleoside triphosphates, and was insensitive to inhibitors of DNA-dependent RNA polymerases. The 32P-labeled enzyme reaction products were largely dsRNAs as indicated by resistance to S1 nuclease and RNase A at high but not low ionic strength. The in vitro synthesized dsRNAs hybridized specifically with CarCV genomic dsRNAs, and the radioactive products present in the polymerase reaction mixture sedimented with the virus particles in sucrose density gradients. The data suggest that the RNA-dependent RNA polymerase associated with CarCV particles is a replicase which catalyzes the synthesis of copies of the genomic dsRNAs.