Silencing of ATP Synthase β Impairs Egg Development in the Leafhopper Scaphoideus titanus, Vector of the Phytoplasma Associated with Grapevine Flavescence Dorée

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 in S. titanus transcriptome 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 leafhopper Euscelidiusvariegatus previously showed female sterility and lack of mature eggs in ovaries. Here, alteration of developing egg morphology in S. titanus ovaries 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.variegatus was 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 in E. variegatus, indicating that off-target effects must be evaluated case by case. The effectiveness of RNAi in S. titanus provides 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.

Scaphoideus titanus Ball feeding behaviour on three grapevine cultivars with different susceptibilities to Flavescence dorée

Scaphoideus titanus (Ball) is a grapevine-feeder leafhopper, and the most important vector of Flavescence dorée of grapevine (FD), a disease associated with phytoplasmas belonging to ribosomal subgroups 16Sr-V–C and –D. FD is a major constraint to viticulture in several European countries and, so far, its control has relied on roguing of infected plants and insecticide applications against the vector. Detailed knowledge on different levels of the multifaceted phytoplasma-plant-vector relationship is required to envisage and explore more sustainable ways to control the disease spread. In the present work, S. titanus feeding behaviour was described on three grapevine cultivars: Barbera (susceptible to FD), Brachetto, and Moscato (tolerant to FD) using the Electrical Penetration Graph (EPG) technique. Interestingly, no differences were highlighted in the non-phloem probing phases, thus suggesting that the tested cultivars have no major differences in the biochemical composition or structure of the leaf cuticle, epidermis or mesophyll, that can affect the first feeding phases. On the contrary, the results showed significant differences in leafhopper feeding behaviour in terms of the duration of the phloem feeding phase, longer on Barbera and shorter on Brachetto and Moscato, and of the frequency of interruption-salivation events inside the phloem, higher on Brachetto and Moscato. These findings indicate a preference for the Barbera variety, that appears a more suitable hosts for the leafhopper. Scaphoideus titanus feeding behaviour on Barbera correlates with an enhanced FDp transmission efficiency, thus explaining, at least in part, the higher susceptibility of this variety to FD. The mechanisms for the non-preference for Brachetto and Moscato are discussed, and a possible antixenosis is hypothesized. We propose that breeding for resistance against FD should take into account both plant traits associated with the response to the phytoplasmas and to the vector.

Koliko znamo o američkom cvrčku (Scaphoideus titanus Ball, 1932)?

Američki cvrčak (Scaphoideus titanus Ball, 1932) jedan je od najznačajnijih vektora zlatne žutice vinove loze. Zakonskim regulativama pokušava se vektora staviti pod nadzor, a poljoprivredni proizvođači dužni su provoditi mjere kako bi spriječili epidemijsko širenje bolesti. Prvi stadiji ličinke jako su sitni što proizvođačima otežava njihovo rano otkrivanje i određivanje roka za zaštitu od ovog štetnika. Kako bi pravovremeno primijenili zaštitu potrebno je pratiti pojavu štetnika jer njegov razvoj najviše ovisi o vremenskim uvjetima. Za suzbijanje američkog cvrčka uglavnom se primjenjuje kemijska zaštita. Jako je bitno paziti i na ostali biljni sastav u vinogradu te izbjeći druge biljke domaćine koje mogu poslužiti štetniku za održavanje. Preporuka je i provođenje edukacija u polju za vinogradare o biologiji ovog štetnika.

Interactions between the flavescence dorée phytoplasma and its insect vector indicate lectin-type adhesion mediated by the adhesin VmpA

The flavescence dorée phytoplasma undergoes a propagative cycle in its insect vectors by first interacting with the insect cell surfaces, primarily in the midgut lumen and subsequently in the salivary glands. Adhesion of flavescence dorée phytoplasma to insect cells is mediated by the adhesin VmpA. We hypothesize that VmpA may have lectin-like activity, similar to several adhesins of bacteria that invade the insect gut. We first demonstrated that the luminal surface of the midgut and the basal surface of the salivary gland cells of the natural vector Scaphoideus titanus and those of the experimental vector Euscelidius variegatus were differentially glycosylated. Using ELISA, inhibition and competitive adhesion assays, and protein overlay assays in the Euva-6 insect cell line, we showed that the protein VmpA binds insect proteins in a lectin-like manner. In conclusion, the results of this study indicate that N-acetylglucosamine and mannose present on the surfaces of the midgut and salivary glands serve as recognition sites for the phytoplasma adhesin VmpA.

First report of Flavescence Dorée-Related Phytoplasma in a Productive Vineyard in Germany

Flavescence dorée (FD) and Bois noir (BN) are the principal grapevine yellows in Europe caused by distinct phytoplasmas: BN by Candidatus Phytoplasma solani, FD by 16SrV-C and -D phytoplasmas (FDp) transmitted by the introduced Nearctic Deltocephalinae Scaphoideus titanus. FDp is listed as a quarantine pest in the European Union (Regulation (EU) 2019/2072). Black Alder (Alnus glutinosa) is a common asymptomatic host of 16SrV phytoplasmas in Europe and considered the original host of FDp (Malembic-Maher et al. 2020). Palatinate grapevine yellows (PGY) transmitted from alder to grapevine by the Macropsinae Oncopsis alni is not transmissible by S. titanus (Malembic-Maher et al. 2020). Germany is considered free from FD in grapevine and from its vector. A single case in a nursery in 2014 was eradicated (EPPO 2017), and FD was never before detected in a vineyard. Since S. titanus appeared in 2016 in the neighboring French Region of Alsace, monitoring of FD was carried out in Germany following a risk based strategy. It was focused on vineyard plots within a distance of 100 m from stands of alder. A geodata-based risk map (Jalke 2020) was used to localize those plots. All symptomatic vines sampled until September 2020 proved to be infected by BN or, occasionally, by PGY. Eight vines with typical symptoms were sampled in vineyards adjacent to alder stands in the winegrowing region of Rheinhessen in September 2020. Symptoms comprised leaf rolling and discoloration, incomplete lignification, and black pustules arranged in lines along the shoots. Diseased shoots were black and necrotic in December. Leaf midribs were sampled for total nucleic acids extraction. The phytoplasma 16S rRNA gene was amplified by generic primers R16F2/R2-mod followed by a nested PCR using 16Sr(V) group-specific primers R16(V)F1/R1, and primers R16(I)F1/R1 (Lee et al. 1995) to detect ‘Candidatus Phytoplasma solani’, associated with BN. While BN was detected in seven vines, one sample tested positive for 16SrV phytoplasma. This result was confirmed by triplex real-time Taq-Man assay based on rpl14 gene sequences (IPADLAB), by multiplex real-time PCR of map locus as well as by Loop-mediated isothermal amplification (LAMP) according to the EPPO diagnostic standard PM 7/079(2) (EPPO 2016). PCR-products of the map and the vmpA genes (Malembic-Maher et al. 2020) were sequenced and compared to reference sequences to distinguish between FD- and non-FD genotypes. The isolate from the diseased vine (GenBank MW 727272) exhibited 100% identity with map-M38 (GenBank LT221933), a genotype of the map-FD2 cluster. The same genotype was detected in A. glutinosa and Allygus spp. sampled at the infested site. A 234 bp sequence of the first repeat of the vmpA gene (GenBank MW727273) showed 100% identity with the homologous part of isolate FD-92 (GenBank LN680870) of the vmpA-II cluster. It can be concluded, that the symptomatic grapevine was infected by FD and not PGY This is the first report of FD in a productive vineyard in Germany. The infected vine of cv. Silvaner was 25 years old. While infected planting material is an unlikely source of the infection, a transmission of FDp from alder is highly probable. Finding a single FD-infection after several years of testing implies a low risk originating from the wild compartment, but the approach and possible establishment of S. titanus expected to be able to colonize the area (Jeger et al. 2016) justifies further monitoring activities. The infected vine was eradicated.

Climate change in the vineyard: perspectives for pest species in the region of Neuchatel

<p>Global warming increases the need for local climatic studies in wine-producing areas. Winegrowers have to develop strategies to adapt their activities to new climatic conditions and to their various effects on vine culture. Among them, distribution and population dynamics of pest species are likely to change. New species could reach the temperate regions, and some native species could create more damages than previously in the vineyards. In Western Europe, the distribution of the American grapevine leafhopper Scaphoideus titanus has been observed to shift northwards during the last decades (Boudon and Maixner 2007). Plurivoltin species such as the European grapevine moth Lobesia botrana could produce more generations per year (Gutierrez et al. 2018), creating potentially more damages on grapes. To help winegrowers, it is crucial to lead research at local scale, taking into account microclimatic specificities of the vineyards (Mozell and Thach 2014).</p><p> </p><p>In this study, we examine temperature trends during the growing season in the region of Neuchatel and their potential impacts on major vine pest species. We focus on the American grapevine leafhopper and on the European grapevine moth. The American grapevine leafhopper is already established in the Lake Geneva area and could soon reach the Neuchatel area, while the European grapevine moth is already present in the Neuchatel vineyard. We use temperature data over the last 40 years (1980-2019) and two climatic scenarios to assess present suitability for pest development and the perspectives for the next decades.</p><p> </p><p> </p><p>REFERENCES</p><p>Boudon, E. & M. Maixner. 2007. Potential effects of climate change on distribution and activity of insect vectors of grapevine pathogens. In International and multi-disciplinary" Global warming, which potential impacts on the vineyards?".</p><p>Gutierrez, A. P., L. Ponti, G. Gilioli & J. Baumgärtner (2018) Climate warming effects on grape and grapevine moth (Lobesia botrana) in the Palearctic region. Agricultural and Forest Entomology, 20<strong>,</strong> 255-271.</p><p>Mozell, M. R. & L. Thach (2014) The impact of climate change on the global wine industry: Challenges & solutions. Wine Economics and Policy, 3<strong>,</strong> 81-89.</p><p> </p>

Models Applied to Grapevine Pests: A Review

This paper reviews the existing predictive models concerning insects and mites harmful to grapevine. A brief conceptual description is given on the definition of a model and about different types of models: deterministic vs. stochastics, continuous vs. discrete, analytical vs. computer-based, and descriptive vs. data-driven. The main biological aspects of grapevine pests covered by different types of models are phenology, population growth and dynamics, species distribution, and invasion risk. A particular emphasis is put on forecasting epidemics of plant disease agents transmitted by insects with sucking-piercing mouthparts. The most investigated species or groups are the glassy-winged sharpshooter Homalodisca vitripennis (Germar) and other vectors of Xylella fastidiosa subsp. fastidiosa, a bacterium agent of Pierce’s disease; the European grape berry moth, Lobesia botrana (Denis and Schiffermuller); and the leafhopper Scaphoideus titanus Ball, the main vector of phytoplasmas agents of Flavescence dorée. Finally, the present and future of decision-support systems (DSS) in viticulture is discussed.

Recovery from Grapevine Flavescence Dorée in Areas of High Infection Pressure

Flavescence dorée (FD) is a quarantine grapevine disease caused by a phytoplasma transmitted by the leafhopper Scaphoideus titanus Ball. FD management relies on compulsory insecticide treatments, roguing of infected plants, and substitution with certified material. Some grapevine cultivars show a spontaneous remission of symptoms (recovery). To determine if recovery is a suitable strategy to co-exist with disease in areas of strong infestation, the qualitative aspects of grapes, musts, and wines obtained from recovered Barbera and Chardonnay grapevines were investigated in two productive vineyards. Following field observations, about 1500 plants in each vineyard were divided into healthy (asymptomatic and negative in phytoplasma molecular diagnosis) and recovered (asymptomatic the year of observation but infected the year before). Maturation curves and microvinification tests followed by oenological and sensory analyses showed that maturation trends of recovered grapes were in line with those from healthy plants and the final qualities of wines were comparable. The spread of FD has strongly increased in Piedmont (Italy) in recent decades. Management strategies to cope with the disease are necessary to preserve traditional wine production. Despite the yield from recovered grapevines is quantitatively lower than that from healthy ones, we showed here that the wine quality is, however, preserved.