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.

First report of Flavescence dorée-related Phytoplasma in Grapevine in Germany.

Flavescence dorée (FD) and Bois noir (BN) are the principal grapevine yellows diseases in Europe caused by distinct phytoplasmas: FD by 16SrV phytoplasmas (FDp), BN by Candidatus Phytoplasma solani. FDp is spread epidemically by the introduced Nearctic Deltocephalinae Scaphoideus titanus and is listed as a quarantine pest in the European Union (Regulation (EU) 2019/2072). Black Alder (Alnus glutinosa) is a common asymptotic 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 (Maixner et al. 2000) is not transmissible by S. titanus, unlike isolates transmitted by the autochthonous Deltocephalinae Allygus spp. and the invasive Orientus ishidae (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). Since S. titanus was detected in 2016 in the neighboring French Region of Alsace, monitoring of FD was carried out in Germany. It was focused on vineyards within a distance of 100 m from stands of alder trees. A geodata-based risk map (Jalke 2020) was used to identify 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, black pustules on shoots, dried inflorescences and shriveled berries. Diseased shoots were black and necrotic in December. Leaf midribs were sampled for total DNA 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-Gene (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 exhibited 100% identity with map-M38 (Accession No. 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 showed 100% identity with the S. titanus transmitted isolate FD-92 (Accession No. LN680870) of the vmpA-II cluster. It can be concluded, that the 16SrV-isolate detected in a symptomatic grapevine is infected by FD and not PGY. This is the first report of FD in a 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 of the vector S. titanus justifies further monitoring activities. The infected vine was eradicated.

Risk Assessment for the Spread of Flavescence Dorée-Related Phytoplasmas from Alder to Grapevine by Alternative Insect Vectors in Germany.

"Flavescence dorée" (FD)-related phytoplasmas are widespread in alder in Germany and their transmission to grapevine represents a high risk for FD outbreaks when the primary vector, Scaphoideus titanus, becomes present in the future. Therefore, the potential role of the Deltocephalinae leafhopper species in transmitting FD-related phytoplasmas from alder to grapevine was studied in extensive transmission trials conducted between 2017 and 2020. The transmission capacity of autochthonous Allygus spp. and the invasive Orientus ishidae captured on infected alder trees was tested under controlled conditions using various test designs, including grouped insects and single-insect studies. The latter experiments were analyzed in terms of survival probability, transmission success and phytoplasma load in the insects, measured by quantitative PCR. A minimum inoculation titer (MIT) required for successful transmission to alder was defined for both Allygus spp. and O. ishidae. While Allygus spp. exhibited slightly better survival on Vitis vinifera compared to O. ishidae, the latter displayed higher phytoplasma loads and greater transmission success. Although all species were capable of infecting alder seedlings, O. ishidae was able to transmit 16SrV-phytoplasmas directly to single grapevines. Infective adults of O. ishidae were captured from the beginning of July until the end of August, while Allygus spp. were only considered infective towards the end of the season. Thus, O. ishidae likely poses a higher risk for FD transmission from alder to grapevine, albeit at a very low level, as only five out of 90 transmission trials to V. vinifera were successful.

Characteristics of the spread of apple proliferation by its vector Cacopsylla picta.

The distribution and natural phytoplasma infection of Cacopsylla picta were investigated during a long-term field survey between 2002 and 2009 in commercial and abandoned apple proliferation-infected orchards throughout Germany, northern Switzerland, and eastern France. Comparable population dynamics were described for the different sites whereas considerable variations in the absolute population densities were observed among the years. Individual polymerase chain reaction (PCR) testing revealed, for each year, a rather stable natural infection rate with ?Candidatus Phytoplasma mali? of ?10% for overwintered adults of C. picta. Both genders were equally highly infected although more females were caught. The overall male/female ratio was 1:1.5. No direct correlation was found between the infection status of the orchard and the infection rate of overwintered C. picta. No influence of agricultural practices was seen. However, a relationship between the incidence of the disease and the vector population density became evident on a regional scale. Successful transmission of ?Ca. P. mali? occurred each year with overwintered individuals as well as with new adults. The transmission efficiency varied among the years within 8 to 45% for overwintered adults and 2 to 20% for individuals of the new generation. The load of single C. picta with ?Ca. P. mali? was determined by quantitative real-time PCR. High phytoplasma titers were measured in overwintered adults already at their first appearance in the orchards after remigration from their overwintering hosts. Thus, the data indicate the transmission of the disease on a regional scale by remigrant adults of C. picta and at a local scale within the same season by emigrant adults which developed on infected plants.

Cacopsylla melanoneura has no relevance as vector of apple proliferation in Germany.

Long-term field surveys on the distribution and natural infection rates of Cacopsylla melanoneura were carried out in commercial and abandoned apple-proliferation-infected orchards throughout Germany, northern Switzerland, and eastern France. Although the infection rates of some orchards reached up to 80%, only 0.09% of all C. melanoneura collected on apple were infected by the pathogen 'Candidatus Phytoplasma mali'. Despite higher population densities, no infected individual was found on wild hawthorn. Individuals of C. melanoneura were not able to transmit phytoplasmas to healthy plants, and even the acquisition of 'Ca. P. mali' from infected plants was very inefficient. Quantitative real-time polymerase chain reaction demonstrated that the very few infected individuals of C. melanoneura harbored phytoplasma concentrations 10,000 times lower than individuals of C. picta, the main vector species in Germany. Oviposition bioassays showed that hawthorn is the preferred reproduction host plant for C. melanoneura in Germany, not apple. Because hawthorn is not a suitable host plant for 'Ca. P. mali', it does not play a role in the spread of apple proliferation. In contrast to data reported from northwestern Italy, C. melanoneura developed on either apple or hawthorn has no relevance as a vector of apple proliferation in Germany. The existence of epidemiologically different populations is proposed.