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. Since the first outbreaks, Flavescence dorée epidemics had been associated to the introduction of the North American leafhopper vector Scaphoideus titanus. We hereby show that the associated phytoplasma originated from European alders and that epidemics in grapevine are restricted to some phytoplasma genetic variants pre-existing in this wild asymptomatic plant host. The compatibility of this phytoplasma to the introduced S. titanus insect vector resulted from the adaptation of phytoplasma variable membrane proteins Vmps to leafhoppers of the same subfamily living on alders. Vmps organization is similar to adhesion related proteins (ARP) and seems to allow the duplication of pre-adapted repeated domains. This suggests a key role of the Vmp adhesins in the life-style of phytoplasmas infecting woody hosts that rely on the adaptation to new insect vectors to expand their plant-host range.

Grapevine leafroll disease (GLD) is considered one of the most widespread grapevine virus diseases, causing severe economic losses worldwide. To date, six grapevine leafroll-associated viruses (GLRaVs) are known as causal agents of the disease, of which GLRaV-1 and -3 induce the strongest symptoms. Due to the lack of efficient curative treatments in the vineyard, identification of infected plants and subsequent uprooting is crucial to reduce the spread of this disease. Ground-based hyperspectral imaging (400–2500 nm) was used in this study in order to identify white and red grapevine plants infected with GLRaV-1 or -3. Disease detection models have been successfully developed for greenhouse plants discriminating symptomatic, asymptomatic, and healthy plants. Furthermore, field tests conducted over three consecutive years showed high detection rates for symptomatic white and red cultivars, respectively. The most important detection wavelengths were used to simulate a multispectral system that achieved classification accuracies comparable to the hyperspectral approach. Although differentiation of asymptomatic and healthy field-grown grapevines showed promising results further investigations are needed to improve classification accuracy. Symptoms caused by GLRaV-1 and -3 could be differentiated.

Grapevine yellows (GY) are serious phytoplasma-caused diseases affecting viticultural areas worldwide. At present, two principal agents of GY are known to infest grapevines in Germany: Bois noir (BN) and Palatinate grapevine yellows (PGY). Disease management is mostly based on prophylactic measures as there are no curative in-field treatments available. In this context, sensor-based disease detection could be a useful tool for winegrowers. Therefore, hyperspectral imaging (400–2500 nm) was applied to identify phytoplasma-infected greenhouse plants and shoots collected in the field. Disease detection models (Radial-Basis Function Network) have successfully been developed for greenhouse plants of two white grapevine varieties infected with BN and PGY. Differentiation of symptomatic and healthy plants was possible reaching satisfying classification accuracies of up to 96%. However, identification of BN-infected but symptomless vines was difficult and needs further investigation. Regarding shoots collected in the field from different red and white varieties, correct classifications of up to 100% could be reached using a Multi-Layer Perceptron Network for analysis. Thus, hyperspectral imaging seems to be a promising approach for the detection of different GY. Moreover, the 10 most important wavelengths were identified for each disease detection approach, many of which could be found between 400 and 700 nm and in the short-wave infrared region (1585, 2135, and 2300 nm). These wavelengths could be used further to develop multispectral systems.

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.

“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.

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.