Barbara Jarausch

The vineyards of Palatinate (Germany) and Alsace (France) in the Upper Rhine valley are still free from Flavescence dorée (FD) disease, but the main vector in grapevine, the leafhopper Scaphoideus titanus , has recently been detected. Knowledge of reservoir plants and alternative vectors of FD phytoplasmas (FDp) is crucial for management strategies. An interregional survey showed that among 12 main wild woody plants and liana species sampled in the environments of vineyards, only alders were infected by FDp, with high prevalence of MAP-FD2 genotypes. Yellow sticky traps were used to record the abundance and analyze the natural infection rate of potential leafhopper vectors on alders, clematis and cultivated grapevine. Live specimens were collected for inoculation trials. High infection rates and transmission capacities of resident Allygus spp. and the invasive Orientus ishidae were confirmed. In addition, five other Deltocephalinae species were identified as potential vectors of FDp. Lamprotettix nitidulus showed a high abundance and high infection rates with FDp in both regions. Its transmission competence of FDp to alder and broad bean could be demonstrated through repeated trials and high FDp titers were measured in infective individuals. Similar titers were measured in Synophropsis lauri, suggesting it could also transmit FDp. Fieberiella florii , Euscelidius variegatus and Japananus hyalinus may also play a role for the spread of 16SrV group phytoplasmas. Six new MAP-FD1 and FD2 genotypes were detected in leafhoppers. The prevalent and only transmitted genotype was M38: a vectotype II compatible with S. titanus transmission. The results show that the natural spread of FDp among alders is more complex than previously thought, and thus, the risk of transmissions to grapevine is more difficult to evaluate.

Pentastiridius leporinus (Hemiptera: Fulgoromorpha: Cixiidae) is considered the main vector of three phloem-restricted pathogens, γ-proteobacteria ‘Candidatus Arsenophonus phytopathogenicus’, ‘Candidatus Phytoplasma solani’ (Stolbur phytoplasma), and 16SrXII-P phytoplasma, which infect two economically important crops, sugar beet and potato. In this study, four pop-up tents (1 m²) were set up in a carrot (Daucus carota L.) field located in Bingen on the Rhine to determine whether egg laying, development of nymphs, and infection is possible on this vegetable. We further show that the planthopper P. leporinus can complete significant parts of its life cycle on carrots in the field, suggesting that it has expanded its host range to this crop in addition to sugar beet and potato. In addition, this study shows that P. leporinus is able to transmit phloem-restricted pathogens to carrots.

“Flavescence dorée” (FD) is a threatening disease of grapevine associated with phytoplasmas of the 16SrV ribosomal group and is epidemically spread within vineyards by the Nearctic leafhopper Scaphoideus titanus. However, recent studies demonstrated that the spread of FD is not only related to this pathosystem but may include alternative vectors and reservoir plants. Alders have been shown harbouring different 16SrV-strains that can be transmitted from alder to alder by Orientus ishidae and Allygus spp., and by O. ishidaealso from alder to grapevine. In all cases the Map-FD2 strain M38 was transmitted. Therefore, a monitoring for both leafhopper species collected on alders has been conducted over 5 years in different wine-growing regions in Germany to investigate their occurrence and infection status. O. ishidae individuals naturally infected with strain M38 were captured in all regions except Mosel. Allygus spp. were also infected with strain M38 but harboured in addition several other non-epidemic strains. Both species captured in Württemberg were able to transmit strain M38 to alder test plants. The results indicate that the widespread occurrence of O. ishidae infected with the epidemic “flavescence dorée” strain M38 represents a risk for the spread of “flavescence dorée” in regions that are so far free from FD.

In the frame of a national survey program, a monitoring for the presence of Scaphoideus titanus was conducted in the southern part of Baden-Württemberg, Germany. Yellow sticky traps were installed in different vineyards in a zone of about 20 km north to south close to the Rhine Valley from July until October 2024 with a two-week change. At the beginning of August, the first specimens were detected and identified as S. titanusand until the end of September, about 500 further S. titanus were collected on the traps. In total, 436 individuals were analysed for phytoplasma infection with different molecular methods. So far, no “flavescence dorée” phytoplasma presence could be confirmed. However, some suspicious cases made a reliable diagnostic difficult. Therefore, a quantitative PCR assay was developed to check the quality of S. titanus DNA extraction.

Apple proliferation (AP) is an economically important disease in many apple-growing regions caused by ‘Candidatus Phytoplasma mali’ which is spread by migrating psyllid vectors on a regional scale. As infected trees in orchards are the only inoculum source, the early eradication of those trees is one of the most efficient strategies to prevent further spread of AP. Remote sensing is a promising rapid and cost-effective tool to identify infected trees on a regional scale. AP-induced premature leaf reddening was evaluated as a reliable symptom for remote sensing by monitoring more than 20,000 trees in 68 different orchards with 20 representative cultivars from 2019 to 2022 in a highly AP-affected region in Southwest Germany. Specific AP symptoms were almost 100% correlated with molecular detection of ‘Ca. P. mali’ and these specific symptoms were almost 100% correlated with leaf reddening. ‘Ca. P. mali’ was detected in 71–97% of trees which showed partial or entire reddening without any other AP symptom. Experimental and field data showed that reddening was induced by cold night and warm day temperatures (about 5 °C vs. 20 °C) in September. Quantification of the phytoplasma by real-time PCR showed no correlation with the intensity of reddening in the leaf. PCR-RFLP subtyping revealed no influence of different ‘Ca. P. mali’ strains on the symptom expression. In conclusion, leaf reddening in late September/early October was a reliable symptom useful for remote sensing of AP.

“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 doree” (FD) and “bois noir” (BN) are the most important phytoplasma diseases of grapevine. While BN is widespread in German winegrowing regions, only one single grapevine plant infected with FD phytoplasma has been reported and eradicated in 2020. Although the main vector of FD, Scaphoideus titanus, is not present in Germany, the FD phytoplasma is classified as quarantine pest because of its epidemic potential and a systematic monitoring of the disease and the vector is mandatory in Germany. Furthermore, a non-epidemic strain Palatinate grapevine yellows (PGY), with the same symptomatology as FD and BN is present in Germany. For large scale monitoring digital multilevel monitoring systems for grapevine yellows (GY) are being developed. Reference plots with infection by either BN or PGY in risk zones in southwestern Germany and Wurttemberg have been visually monitored and symptomatic grapevines were analysed by molecular means. In parallel, the same samples were examined with different sensor-based methods to define a correlation between phytoplasma presence and specific spectral signatures. A spectral distinction to similar symptoms due to virus infection, insect damage, and nutritional deficiencies was investigated for the system validation.

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.

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.