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Scaphoideus titanus(Hemiptera:Cicadellidae) is the natural vector of Flavescence dorée phytoplasma, a quarantine pest of grapevine with severe impact on European viticulture. RNA interference (RNAi) machinery components are present inS. titanustranscriptome and injection of ATP synthase β dsRNAs into adults caused gene silencing, starting three days post injection (dpi) up to 20 dpi, leading to decrease cognate protein. Silencing of this gene in the closely related leafhopperEuscelidiusvariegatuspreviously showed female sterility and lack of mature eggs in ovaries. Here, alteration of developing egg morphology inS. titanusovaries as well as overexpression of hexamerin transcript (amino acid storage protein) and cathepsin L protein (lysosome proteinase) were observed in dsATP-injected females. To evaluate RNAi-specificity,E.variegatuswas used as dsRNA-receiving model-species. Different doses of two sets of dsRNA-constructs targeting distinct portions of ATP synthase β gene of both species induced silencing, lack of egg development, and female sterility inE. variegatus, indicating that off-target effects must be evaluated case by case. The effectiveness of RNAi inS. titanusprovides a powerful tool for functional genomics of this non-model species and paves the way toward RNAi-based strategies to limit vector population, despite several technical and regulatory constraints that still need to be overcome to allow open field application.

p { margin-bottom: 0.1in; direction: ltr; color: rgb(0, 0, 0); line-height: 120%; }p.western { font-family: "Calibri", sans-serif; font-size: 11pt; }p.cjk { font-family: "Calibri", sans-serif; font-size: 11pt; }p.ctl { font-family: "Calibri", sans-serif; font-size: 11pt; }a:link { color: rgb(0, 0, 255); } In these study, gene silencing techniques mediated by RNA interference (RNAi) are applied to the leafhopper Euscelidius variegatus, a natural vector of chrysanthemum yellows phytoplasma and an efficient vector of Flavescence dorée phytoplasma under laboratory conditions. Phytoplasmas are plant pathogenic bacteria transmitted by sap-feeding insects belonging to the order Hemiptera and associated with many diseases that cause severe economic impacts on many crops worldwide. The purpose of the work is to shed light on insect genes potentially involved in the interaction with phytoplasmas, and, more specifically, in the acquisition and transmission mechanisms of these pathogens. In particular, the ability of actin and ATP synthase β dsRNAs (double stranded RNAs) to induce RNAi effects after abdominal injection is being tested. Both genes are known to be involved in the interaction between phytoplasmas and their insect hosts, so an efficient perturbation of their expression is likely to cause a modification in the epidemiological cycle of this plant disease. The possibility of using insect viruses as dsRNA delivery tools (virus induced gene silencing) is also explored. During a RNA-seq project of E. variegatus, a contig with high sequence similarity to known picorna-like viruses was in fact identified among the assembled insect transcripts. We are currently constructing an infectious clone of this virus, named Euscelidius variegatus virus 1 (EVV-1), which would represent a groundbreaking tool to be used directly as a biocontrol agent, providing additional tools to integrate or even replace insecticide treatments towards a more sustainable agriculture.

Acibenzolar-S-methyl (BTH), a functional analogue of salicylic acid (SA), is known to elicit a systemic resistance across a broad range of plant-pathogen interactions, but so far it has not been tested against flavescence dorée (FDP), one of the most devastating grapevine diseases. The aim of this work was to evaluate the activity of BTH in preventing FDP transmission by the insect vector and in inducing recovery of infected grapevines.Repeated 2 mM applications of BTH to test grapevine cuttings (cv. Barbera) exposed to adults of the infectious vector Scaphoideus titanus Ball reduced the rate of infected plants. The effect was not recorded following similar BTH applications to highly susceptible young in vitro propagated vines. A high natural recovery rate (more than 70%) was observed over a 3 year period in field-infected grapevines of the same cultivar. Under these conditions, BTH repeated applications over the whole period clearly failed to increase recovery of field-infected grapevines.Following a 3 year experiment, it can be concluded that, although high doses and repeated applications of BTH reduced vector transmission of FDP, BTH was ineffective in inducing recovery of FDP-infected grapevines cv. Barbera under field conditions. © 2016 Society of Chemical Industry.

Phytoplasma quantification in ‘Transmitter’ and ‘Non-transmitter’ insects. Table indicating mean (± standard error) chrysanthemum yellows phytoplasma (CYP) titer measured in Euscelidius variegatus, following microinjection with CYP suspension or CYP suspension plus antibody A416. (PDF 282 kb)

Phytoplasma quantification in dissected salivary glands. Table indicating mean (± standard error) chrysanthemum yellows phytoplasma (CYP) titer measured by qPCR in thoroughly washed salivary glands of healthy Euscelidius variegatus following incubation with CYP suspension alone or CYP suspension plus antibody A416, according to the protocol developed for the internalization assay. (PDF 178 kb)

Optimization of abdominal microinjection parameters. Description of parameter optimizations for abdominal microinjection experiments. (PDF 56 kb)