Pentastiridius leporinus (Linnaeus, 1761) as a Vector of Phloem-Restricted Pathogens on Potatoes: 'Candidatus Arsenophonus Phytopathogenicus' and 'Candidatus Phytoplasma Solani'.

In Germany, the phloem-sucking planthopper Pentastiridius leporinus (Hemiptera: Cixiidae) currently represents the epidemiological driver for the spread of the syndrome "Basses Richesses" in sugar beets, which results in a reduced sugar content and an economic loss for the farmers. This disease is associated with the γ-proteobacterium 'Candidatus Arsenophonus phytopathogenicus' and the Stolbur phytoplasma 'Candidatus Phytoplasma solani'. Recently, P. leporinus was found in potato fields in Germany and is associated with Stolbur-like symptoms in this crop. In this study, we confirmed that the vector completes its lifecycle on sugar beets as well as on potatoes when reared under controlled conditions. Transmission experiments with adults of this vector combined with molecular analyses showed, for the first time, that both pathogens are transmitted by this vector to potatoes. For an accurate assessment of the Ca. P. solani and Ca. A. phytopathogenicus titers in the vector and host plants, gBlocks derived from the hsp20- and 16S rRNA genes were employed, respectively. For Ca. P. solani, the limit of detection was determined in potato and sugar beet material. The results of this study will further the research on the epidemiology of the syndrome "Basses Richesses" and "Stolbur" diseases and the response of host plants and vector insects to both pathogens.

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.

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.

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.

Survival relative to new and ancestral host plants, phytoplasma infection, and genetic constitution in host races of a polyphagous insect disease vector.

Dissemination of vectorborne diseases depends strongly on the vector's host range and the pathogen's reservoir range. Because vectors interact with pathogens, the direction and strength of a vector's host shift is vital for understanding epidemiology and is embedded in the framework of ecological specialization. This study investigates survival in host-race evolution of a polyphagous insect disease vector, Hyalesthes obsoletus, whether survival is related to the direction of the host shift (from field bindweed to stinging nettle), the interaction with plant-specific strains of obligate vectored pathogens/symbionts (stolbur phytoplasma), and whether survival is related to genetic differentiation between the host races. We used a twice repeated, identical nested experimental design to study survival of the vector on alternative hosts and relative to infection status. Survival was tested with Kaplan-Meier analyses, while genetic differentiation between vector populations was quantified with microsatellite allele frequencies. We found significant direct effects of host plant (reduced survival on wrong hosts) and sex (males survive longer than females) in both host races and relative effects of host (nettle animals more affected than bindweed animals) and sex (males more affected than females). Survival of bindweed animals was significantly higher on symptomatic than nonsymptomatic field bindweed, but in the second experiment only. Infection potentially had a positive effect on survival in nettle animals but due to low infection rates the results remain suggestive. Genetic differentiation was not related to survival. Greater negative plant-transfer effect but no negative effect of stolbur in the derived host race suggests preadaptation to the new pathogen/symbiont strain before strong diversifying selection during the specialization process. Physiological maladaptation or failure to accept the ancestral plant will have similar consequences, namely positive assortative mating within host races and a reduction in the likelihood of oviposition on the alternative plant and thus the acquisition of alternative stolbur strains.

Sympatric diversification vs. immigration: deciphering host-plant specialization in a polyphagous insect, the stolbur phytoplasma vector Hyalesthes obsoletus (Cixiidae).

The epidemiology of vector transmitted plant diseases is highly influenced by dispersal and the host-plant range of the vector. Widening the vector's host range may increase transmission potential, whereas specialization may induce specific disease cycles. The process leading to a vector's host shift and its epidemiological outcome is therefore embedded in the frameworks of sympatric evolution vs. immigration of preadapted populations. In this study, we analyse whether a host shift of the stolbur phytoplasma vector, Hyalesthes obsoletus from field bindweed to stinging nettle in its northern distribution range evolved sympatrically or by immigration. The exploitation of stinging nettle has led to outbreaks of the grapevine disease bois noir caused by a stinging nettle-specific phytoplasma strain. Microsatellite data from populations from northern and ancestral ranges provide strong evidence for sympatric host-race evolution in the northern range: Host-plant associated populations were significantly differentiated among syntopic sites (0.054 < F(HT) < 0.098) and constant over 5 years. While gene flow was asymmetric from the old into the predicted new host race, which had significantly reduced genetic diversity, the genetic identity between syntopic host-race populations in the northern range was higher than between these populations and syntopic populations in ancestral ranges, where there was no evidence for genetic host races. Although immigration was detected in the northern field bindweed population, it cannot explain host-race diversification but suggests the introduction of a stinging nettle-specific phytoplasma strain by plant-unspecific vectors. The evolution of host races in the northern range has led to specific vector-based bois noir disease cycles.

'Candidatus Phytoplasma convolvuli', a new phytoplasma taxon associated with bindweed yellows in four European countries.

Plants of Convolvulus arvensis exhibiting symptoms of undersized leaves, shoot proliferation and yellowing, collectively defined as bindweed yellows, were sampled in different regions of Europe and assessed for phytoplasma infection by PCR amplification using phytoplasma universal rRNA operon primer pairs. Positive results were obtained for all diseased plants. RFLP analysis of amplicons comprising the16S rRNA gene alone or the16S rRNA gene and 16-23S intergenic spacer region indicated that the detected phytoplasmas were distinguishable from all other previously described rRNA gene sequences. Analysis of 16S rRNA gene sequences derived from seven selected phytoplasma strains (BY-S57/11, BY-S62/11, BY-I1015, BY-I1016, BY-BH1, BY-BH2 and BY-G) showed that they were nearly identical (99.9-100% gene sequence similarity) but shared less than 97.5% similarity with comparable sequences of other phytoplasmas. Thus, BY phytoplasmas represent a new taxon whose closest relatives are stolbur phytoplasma strains and 'Candidatus Phytoplasma fragariae' with which they share 97.2% and 97.1% 16S rRNA gene sequence similarity, respectively. Phylogenetic analysis of 16S rRNA gene sequences confirmed that bindweed yellows phytoplasma strains collectively represent a distinct lineage within the phytoplasma clade and share a common ancestor with previously published or proposed 'Candidatus Phytoplasma' taxa within a major branch including aster yellows and stolbur phytoplasmas. On the basis of unique 16S rRNA gene sequences and biological properties that include a single host plant species and a geographical distribution limited to parts of Europe, the bindweed yellows (BY) phytoplasmas represent a coherent but discrete taxon, 'Candidatus Phytoplasma convolvuli', with strain BY-S57/11 (GenBank accession no. JN833705) as the reference strain.

Impact of vector dispersal and host-plant fidelity on the dissemination of an emerging plant pathogen.

Dissemination of vector-transmitted pathogens depend on the survival and dispersal of the vector and the vector's ability to transmit the pathogen, while the host range of vector and pathogen determine the breath of transmission possibilities. In this study, we address how the interaction between dispersal and plant fidelities of a pathogen (stolbur phytoplasma tuf-a) and its vector (Hyalesthes obsoletus: Cixiidae) affect the emergence of the pathogen. Using genetic markers, we analysed the geographic origin and range expansion of both organisms in Western Europe and, specifically, whether the pathogen's dissemination in the northern range is caused by resident vectors widening their host-plant use from field bindweed to stinging nettle, and subsequent host specialisation. We found evidence for common origins of pathogen and vector south of the European Alps. Genetic patterns in vector populations show signals of secondary range expansion in Western Europe leading to dissemination of tuf-a pathogens, which might be newly acquired and of hybrid origin. Hence, the emergence of stolbur tuf-a in the northern range was explained by secondary immigration of vectors carrying stinging nettle-specialised tuf-a, not by widening the host-plant spectrum of resident vectors with pathogen transmission from field bindweed to stinging nettle nor by primary co-migration from the resident vector's historical area of origin. The introduction of tuf-a to stinging nettle in the northern range was therefore independent of vector's host-plant specialisation but the rapid pathogen dissemination depended on the vector's host shift, whereas the general dissemination elsewhere was linked to plant specialisation of the pathogen but not of the vector.

Multilocus sequence typing confirms the close genetic interrelatedness of three distinct flavescence dorée phytoplasma strain clusters and group 16SrV phytoplasmas infecting grapevine and alder in Europe.

Vineyards of southern France and northern Italy are affected by the flavescence dorée (FD) phytoplasma, a quarantine pathogen transmitted by the leafhopper of Nearctic origin Scaphoideus titanus. To better trace propagation of FD strains and identify possible passage between the vineyard and wild plant compartments, molecular typing of phytoplasma strains was applied. The sequences of the two genetic loci map and uvrB-degV, along with the sequence of the secY gene, were determined among a collection of FD and FD-related phytoplasmas infecting grapevine, alder, elm, blackberry, and Spanish broom in Europe. Sequence comparisons and phylogenetic analyses consistently indicated the existence of three FD phytoplasma strain clusters. Strain cluster FD1 (comprising isolate FD70) displayed low variability and represented 17% of the disease cases in the French vineyard, with a higher incidence of the cases in southwestern France. Strain cluster FD2 (comprising isolates FD92 and FD-D) displayed no variability and was detected both in France (83% of the cases) and in Italy, whereas the more-variable strain cluster FD3 (comprising isolate FD-C) was detected only in Italy. The clonal property of FD2 and its wide distribution are consistent with diffusion through propagation of infected-plant material. German Palatinate grapevine yellows phytoplasmas (PGY) appeared variable and were often related to some of the alder phytoplasmas (AldY) detected in Italy and France. Finally, phylogenetic analyses concluded that FD, PGY, and AldY were members of the same phylogenetic subclade, which may have originated in Europe.