Genetic Diversity of Flavescence Dorée Phytoplasmas at the Vineyard Scale.

To study the role of wild areas around the vineyards in the epidemiology of flavescence dorée (FD) and track the origin of new foci, two phytoplasma genetic markers, dnaK and malG, were developed for FD phytoplasma (FDp) characterization. The two genes and the vmpA locus were used to genetically characterize FDp populations at seven agroecosystems of a wine-growing Italian region. Vitis vinifera, "gone-wild" V. vinifera and rootstocks, Clematis spp., and Scaphoideus titanus adults were sampled within and outside the vineyards. A range of genotypes infecting the different hosts of the FDp epidemiological cycle was found. Type FD-C isolates were fairly homogeneous compared to type FD-D ones. Most of the FD-D variability was correlated with the malG sequence, and a duplication of this locus was demonstrated for this strain. Coinfection with FD-C and FD-D strains was rare, suggesting possible competition between the two. Similar levels of FDp genetic variation recorded for grapevines or leafhoppers of cultivated and wild areas and co-occurrence of many FDp genotypes inside and outside the vineyards supported the idea of the importance of wild or abandoned Vitis plants and associated S. titanus insects in the epidemiology of the disease. Genetic profiles of FDp found in Clematis were never found in the other hosts, indicating that this species does not take part in the disease cycle in the area. Due to the robustness of analyses using dnaK for discriminating between FD-C and FD-D strains and the high variability of malG sequences, these are efficient markers to study FDp populations and epidemiology at a small geographical scale.IMPORTANCE Flavescence dorée, a threatening disease of grapevine caused by FD phytoplasma (FDp), is distributed within the most important wine-producing areas of Europe and has severe effects on both vineyard productivity and landscape management. FDp is a quarantine pest in Europe, and despite the efforts to contain the pathogen, the disease is still spreading. In this work, new genetic markers for the fine genetic characterization of FDp at local scale are presented. Our findings improve the knowledge of FDp epidemiological cycle and offer the possibility of tracking the route of the FDp infection. In particular, due to its high genetic variability, one of the newly developed markers could be sufficient to track the origin of new infection foci, either from the wild areas or from nurseries.

Differential gene expression in two grapevine cultivars recovered from "flavescence dorée".

The biological bases of recovery of two grapevine cultivars, Nebbiolo and Barbera, showing different susceptibility and recovery ability to "flavescence dorée" (FD) phytoplasma infection were investigated. The expression over one vegetative season, in FD-recovered and healthy grapevines, of 18 genes involved in defence, hydrogen peroxide and hormone production was verified at two time points. Difference (Δ) between the relative expressions of August and July were calculated for each target gene of both cultivars. The significance of differences among groups assessed by univariate and multivariate statistical methods, and sPLS-DA analyses of the Δ gene expression values, showed that control and recovered grapevines of both cultivars were clearly separated. The Barbera-specific deregulation of defence genes supports a stronger response of this variety, within a general frame of interactions among H2O2, jasmonate and ethylene metabolisms, common to both varieties. This may strengthen the hypothesis that FD-recovered Barbera grapevines modulate transcription of their genes to cope with potential damages associated to the alteration of their oxidative status. Nebbiolo variety would fit into this picture, although with a less intense response, in line with its lower degree of susceptibility and recovery incidence to FD, compared to Barbera. The results evidenced a scenario where plant response to phytoplasma infection is highly affected by climatic and edaphic conditions. Nevertheless, even after several years from the original FD infection, it was still possible to distinguish, at molecular level, control and recovered grapevines of both cultivars by analyzing their overall-season response, rather than that of a single time point.

Identification of putative effector genes and their transcripts in three strains related to 'Candidatus Phytoplasma aurantifolia'.

Molecular mechanisms underlying phytoplasma interactions with host plants are largely unknown. In this study attempts were made to identify effectors of three phytoplasma strains related to 'Ca. P. aurantifolia', crotalaria phyllody (CrP), faba bean phyllody (FBP), and witches' broom disease of lime (WBDL), using information from draft genome of peanut witches' broom phytoplasma. Seven putative effectors were identified in WBDL genome (SAP11, SAP21, Eff64, Eff115, Eff197, Eff211 and EffSAP67), five (SAP11, SAP21, Eff64, Eff99 and Eff197) in CrP and two (SAP11, Eff64) in FBP. No homologs to Eff64, Eff197 and Eff211 in phytoplasmas of other phylogenetic groups were found. SAP11 and Eff64 homologs of 'Ca. P. aurantifolia' strains shared at least 95.9% identity and were detected in the three phytoplasmas, supporting their role within the group. Five of the putative effectors (SAP11, SAP21, Eff64, Eff115, and Eff99) were transcribed from total RNA extracts of periwinkle plants infected with these phytoplasmas. Transcription profiles of selected putative effectors of CrP, FBP and WBDL indicated that SAP11 transcripts were the most abundant in the three phytoplasmas. SAP21 transcript levels were comparable to those of SAP11 for CrP and not measurable for the other phytoplasmas. Eff64 had the lowest transcription level irrespective of sampling date and phytoplasma isolate. Eff115 transcript levels were the highest in WBDL infected plants. This work reports the first sequence information for 14 putative effectors in three strains related to 'Ca. P. aurantifolia', and offers novel insight into the transcription profile of five of them during infection of periwinkle.

Acquisition of Flavescence Dorée Phytoplasma by Scaphoideus titanus Ball from Different Grapevine Varieties.

Flavescence dorée (FD) is a threat for wine production in the vineyard landscape of Piemonte, Langhe-Roero and Monferrato, Italy. Spread of the disease is dependent on complex interactions between insect, plant and phytoplasma. In the Piemonte region, wine production is based on local cultivars. The role of six local grapevine varieties as a source of inoculum for the vector Scaphoideus titanus was investigated. FD phytoplasma (FDP) load was compared among red and white varieties with different susceptibility to FD. Laboratory-reared healthy S. titanus nymphs were caged for acquisition on infected plants to measure phytoplasma acquisition efficiency following feeding on different cultivars. FDP load for Arneis was significantly lower than for other varieties. Acquisition efficiency depended on grapevine variety and on FDP load in the source plants, and there was a positive interaction for acquisition between variety and phytoplasma load. S. titanus acquired FDP with high efficiency from the most susceptible varieties, suggesting that disease diffusion correlates more with vector acquisition efficiency than with FDP load in source grapevines. In conclusion, although acquisition efficiency depends on grapevine variety and on FDP load in the plant, even varieties supporting low FDP multiplication can be highly susceptible and good sources for vector infection, while poorly susceptible varieties may host high phytoplasma loads.

Role of the major antigenic membrane protein in phytoplasma transmission by two insect vector species.

BACKGROUND: Phytoplasmas are bacterial plant pathogens (class Mollicutes), transmitted by phloem feeding leafhoppers, planthoppers and psyllids in a persistent/propagative manner. Transmission of phytoplasmas is under the control of behavioral, environmental and geographical factors, but molecular interactions between membrane proteins of phytoplasma and vectors may also be involved. The aim of the work was to provide experimental evidence that in vivo interaction between phytoplasma antigenic membrane protein (Amp) and vector proteins has a role in the transmission process. In doing so, we also investigated the topology of the interaction at the gut epithelium and at the salivary glands, the two barriers encountered by the phytoplasma during vector colonization. METHODS: Experiments were performed on the 'Candidatus Phytoplasma asteris' chrysanthemum yellows strain (CYP), and the two leafhopper vectors Macrosteles quadripunctulatus Kirschbaum and Euscelidius variegatus Kirschbaum. To specifically address the interaction of CYP Amp at the gut epithelium barrier, insects were artificially fed with media containing either the recombinant phytoplasma protein Amp, or the antibody (A416) or both, and transmission, acquisition and inoculation efficiencies were measured. An abdominal microinjection protocol was employed to specifically address the interaction of CYP Amp at the salivary gland barrier. Phytoplasma suspension was added with Amp or A416 or both, injected into healthy E. variegatus adults and then infection and inoculation efficiencies were measured. An internalization assay was developed, consisting of dissected salivary glands from healthy E. variegatus exposed to phytoplasma suspension alone or together with A416 antibody. The organs were then either observed in confocal microscopy or subjected to DNA extraction and phytoplasma quantification by qPCR, to visualize and quantify possible differences among treatments in localization/presence/number of CYP cells. RESULTS: Artificial feeding and abdominal microinjection protocols were developed to address the two barriers separately. The in vivo interactions between Amp of 'Candidatus Phytoplasma asteris' Chrysanthemum yellows strain (CYP) and vector proteins were studied by evaluating their effects on phytoplasma transmission by Euscelidius variegatus and Macrosteles quadripunctulatus leafhoppers. An internalization assay was developed, consisting of dissected salivary glands from healthy E. variegatus exposed to phytoplasma suspension alone or together with anti-Amp antibody. To visualize possible differences among treatments in localization/presence of CYP cells, the organs were observed in confocal microscopy. Pre-feeding of E. variegatus and M. quadripunctulatus on anti-Amp antibody resulted in a significant decrease of acquisition efficiencies in both species. Inoculation efficiency of microinjected E. variegatus with CYP suspension and anti-Amp antibody was significantly reduced compared to that of the control with phytoplasma suspension only. The possibility that this was due to reduced infection efficiency or antibody-mediated inhibition of phytoplasma multiplication was ruled out. These results provided the first indirect proof of the role of Amp in the transmission process. CONCLUSION: Protocols were developed to assess the in vivo role of the phytoplasma native major antigenic membrane protein in two phases of the vector transmission process: movement through the midgut epithelium and colonization of the salivary glands. These methods will be useful also to characterize other phytoplasma-vector combinations. Results indicated for the first time that native CYP Amp is involved in vivo in specific crossing of the gut epithelium and salivary gland colonization during early phases of vector infection.

RNA-Seq profile of flavescence dorée phytoplasma in grapevine.

BACKGROUND: The phytoplasma-borne disease flavescence dorée is still a threat to European viticulture, despite mandatory control measures and prophylaxis against the leafhopper vector. Given the economic importance of grapevine, it is essential to find alternative strategies to contain the spread, in order to possibly reduce the current use of harmful insecticides. Further studies of the pathogen, the vector and the mechanisms of phytoplasma-host interactions could improve our understanding of the disease. In this work, RNA-Seq technology followed by three de novo assembly strategies was used to provide the first comprehensive transcriptomics landscape of flavescence dorée phytoplasma (FD) infecting field-grown Vitis vinifera leaves. RESULTS: With an average of 8300 FD-mapped reads per library, we assembled 347 sequences, corresponding to 215 annotated genes, and identified 10 previously unannotated genes, 15 polycistronic transcripts and three genes supposedly localized in the gaps of the FD92 draft genome. Furthermore, we improved the annotation of 44 genes with the addition of 5'/3' untranslated regions. Functional classification revealed that the most expressed genes were either related to translation and protein biosynthesis or hypothetical proteins with unknown function. Some of these hypothetical proteins were predicted to be secreted, so they could be bacterial effectors with a potential role in modulating the interaction with the host plant. Interestingly, qRT-PCR validation of the RNA-Seq expression values confirmed that a group II intron represented the FD genomic region with the highest expression during grapevine infection. This mobile element may contribute to the genomic plasticity that is necessary for the phytoplasma to increase its fitness and endorse host-adaptive strategies. CONCLUSIONS: The RNA-Seq technology was successfully applied for the first time to analyse the FD global transcriptome profile during grapevine infection. Our results provided new insights into the transcriptional organization and gene structure of FD. This may represent the starting point for the application of high-throughput sequencing technologies to study differential expression in FD and in other phytoplasmas with an unprecedented resolution.