When a Palearctic bacterium meets a Nearctic insect vector: Genetic and ecological insights into the emergence of the grapevine Flavescence dorée epidemics in Europe.

Flavescence dorée (FD) is a European quarantine grapevine disease transmitted by the Deltocephalinae leafhopper Scaphoideus titanus. Whereas this vector had been introduced from North America, the possible European origin of FD phytoplasma needed to be challenged and correlated with ecological and genetic drivers of FD emergence. For that purpose, a survey of genetic diversity of these phytoplasmas in grapevines, S. titanus, black alders, alder leafhoppers and clematis were conducted in five European countries. Out of 132 map genotypes, only 11 were associated to FD outbreaks, three were detected in clematis, whereas 127 were detected in alder trees, alder leafhoppers or in grapevines out of FD outbreaks. Most of the alder trees were found infected, including 8% with FD genotypes M6, M38 and M50, also present in alders neighboring FD-free vineyards and vineyard-free areas. The Macropsinae Oncopsis alni could transmit genotypes unable to achieve transmission by S. titanus, while the Deltocephalinae Allygus spp. and Orientus ishidae transmitted M38 and M50 that proved to be compatible with S. titanus. Variability of vmpA and vmpB adhesin-like genes clearly discriminated 3 genetic clusters. Cluster Vmp-I grouped genotypes only transmitted by O. alni, while clusters Vmp-II and -III grouped genotypes transmitted by Deltocephalinae leafhoppers. Interestingly, adhesin repeated domains evolved independently in cluster Vmp-I, whereas in clusters Vmp-II and-III showed recent duplications. Latex beads coated with various ratio of VmpA of clusters II and I, showed that cluster II VmpA promoted enhanced adhesion to the Deltocephalinae Euscelidius variegatus epithelial cells and were better retained in both E. variegatus and S. titanus midguts. Our data demonstrate that most FD phytoplasmas are endemic to European alders. Their emergence as grapevine epidemic pathogens appeared restricted to some genetic variants pre-existing in alders, whose compatibility to S. titanus correlates with different vmp gene sequences and VmpA binding properties.

Characterization by deep sequencing of divergent plum bark necrosis stem pitting-associated virus (PBNSPaV) isolates and development of a broad-spectrum PBNSPaV detection assay.

Plum bark necrosis stem pitting-associated virus (PBNSPaV), the causal agent of plum bark necrosis stem pitting disease, belongs to the genus Ampelovirus in the family Closteroviridae. The complete genome sequence of PBNSPaV isolates from four Prunus sources was determined by pyrosequencing. All isolates showed the same genomic organization as the PBNSPaV reference isolate. The least divergent isolate, found in a peach tree from China, showed an overall 91.8% of nucleotide identity with the type isolate. Two closely related isolates, defining a second cluster of diversity, were found in two Japanese plum lines from France and showed only 82.8% identity with the type isolate. On the other hand, they were highly homologous with two recently described PBNSPaV divergent isolates from China. The fourth and most divergent isolate, from a Chinese peach, showed only 71.2% identity to other PBNSPaV isolates and was not detected by currently available PBNSPaV reverse-transcription polymerase chain reaction detection assays. Complete sequencing of the divergent isolates allowed the development of a more broad-spectrum detection test targeting a conserved region in the P61 gene. Taken together, these results indicate a much broader diversity of PBNSPaV than previously thought and provide for a more robust detection of this still poorly characterized pathogen.

Viral Double-Stranded RNAs (dsRNAs) from Plants: Alternative Nucleic Acid Substrates for High-Throughput Sequencing.

High-throughput sequencing (or next-generation sequencing-NGS) is an emerging technology that allows the detection of plant viruses without any prior knowledge. Various sequencing techniques and various templates can be used as substrate for NGS. This chapter describes an optimized protocol for the extraction of double-stranded RNAs (dsRNAs) from a wide range of plants and for their random amplification prior to NGS sequencing.

Evaluation of the association of nine Helicobacter pylori virulence factors with strains involved in low-grade gastric mucosa-associated lymphoid tissue lymphoma.

Helicobacter pylori has been associated with the development of two malignant diseases: gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Although the cag pathogenicity island, especially the cagA gene, has been linked with adenocarcinoma, few data concerning H. pylori pathogenic factors involved in low-grade gastric MALT lymphoma are available. The goal of this study was to analyze the prevalence of and correlation between genes coding for seven H. pylori virulence factors (cagA, cagE, vacA, iceA, babA, hopQ, and oipA) and two novel adhesins (sabA and hopZ) by comparing a collection of 43 H. pylori strains isolated from patients with low-grade gastric MALT lymphoma to 39 strains isolated from age-matched patients with gastritis only. Our results show that taken individually, none of the nine genes tested can be considered associated with MALT strains and allow us to conclude that MALT pathogenesis is not linked with more proinflammatory H. pylori strains. We demonstrated that in patients infected with strains harboring the iceA1 allele, sabA functional status, and hopZ "off" status, the odds of developing a MALT lymphoma were 10 times higher. However, the low prevalence of such strains (10 of 43 MALT strains) renders this triple association a low-sensitivity marker for MALT strains. Our data confirmed that H. pylori virulence factors are correlated with one another. If the involvement of H. pylori in MALT lymphoma is well established, the pathomechanism by which gastric lymphoma occurs remains to be identified.