PurposeThe study aims to develop a method for detecting and discriminating grapevine yellows (GY) diseases, including Flavescence dorée (FD), Bois noir (BN), and Palatinate grapevine yellows (PGY), using hyperspectral imaging (HSI). Specifically, it seeks to address the challenge of symptom misclassification among visually similar diseases, such as distinguishing between GY diseases and other biotic and abiotic stresses.MethodsHyperspectral images of detached leaves from field with GY diseases, Grapevine Leafroll-associated Virus (V), leafhopper (LH) infestation, iron deficiency (Fe), or magnesium deficiency (Mg) were acquired using a mobile platform in the laboratory or in the field. The images were taken in the spectral range of 400-1,000 nm. Classification models were trained on the mean spectra of the leaves to distinguish between different classes.ResultsThe model achieved F1-scores ranging from 54.2% to 96.8% for white cultivars and exceeded 95% for all six classes for black cultivars. When limited to GY classes and nonsymptomatic leaves, the F1-scores were 89.8%, 78.7%, 63.4%, and 53.4% for nonsymptomatic, FD, PGY, and BN, respectively.ConclusionThe study demonstrates the feasibilityof using HSI to detect and discriminate different biotic and abiotic stresses on grapevine leaves, including distinguishing between symptom-similar GY diseases. The developed mobile platform and classification models show promise for largescale monitoring and diagnosis of GY diseases, potentially improving disease management and reducing the risk of symptom misclassification.

The bois noir (BN) disease of grapevines is widespread in German winegrowing regions. It is associated with ‘Candidatus Phytoplasma solani’, which affects not only grapevines but also other wild and cultivated plants. This pathogen has a complex epidemiology including different insect vectors and various host plants. A study was carried out to investigate the genetic variability of ‘Ca. P. solani’ in different winegrowing regions in Germany. Between 2017 and 2023, samples of grapevine, stinging nettle, bindweed, and other herbaceous plants as well as specimens of different planthopper species colonizing viticultural habitats were analyzed for infection with ‘Ca. P. solani’. All positive tested samples were further characterized by multilocus sequence typing (MLST) based on the genes tuf, stamp, secY, and vmp1. The genetic variability was assessed by RFLP analyses of the tuf and vmp1 PCR products, coupled with sequencing of the stamp and secY amplification products. A total of 1274 grapevines, 35 bindweed, and 18 stinging nettle samples were infected with ‘Ca. P. solani’ but also five samples of other weed species. Among the known and putative insect vectors, specimens of Hyalesthes obsoletus, Reptalus spp., and Dictyophara europaea harbored the phytoplasma. In both plants and insects, two genotype combinations were predominantly associated with the classical bindweed and stinging nettle cycle, respectively. The MLST analysis revealed considerable differences between German isolates and data reported from other European regions and new genotype combinations were identified, indicating new host plant–vector associations.

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.

IntroductionFlavescence dorée (FD) is a European quarantine disease of grapevine with high economic impact. The Flavescence dorée phytoplasmas (FDp) are epidemically spread by the leafhopper Scaphoideus titanus, which has been introduced to Europe from North America. The FDp is of European origin and is widely spread in symptomless alder trees. FD outbreaks were related to three different genetic variants of FDp: MAP-FD1, MAP-FD2 and MAP-FD3. A multitude of non-epidemic genotypes exist in mixed infections in alder trees. Germany is so far free of FD but the possibility of the transmission of MAP-FD2 phytoplasmas from alder to grapevine by the invasive secondary vector Orientus ishidae has been demonstrated experimentally and under natural conditions. Thus, FDp-infected alder represent a risk for FD outbreaks.MethodsGeodata-based analyses were employed to identify potential alder sites adjacent to vineyards in the major wine-growing regions of Germany in Rhineland-Palatinate and Baden-Wuerttemberg. Webmaps for Plant Protection Services were established. In addition, molecular screening tools for the detection of MAP-FD1 and MAP-FD2 phytoplasmas in mixed infections in alders were developed.Results and Discussion1089 alder samples from 171 sites in Rhineland-Palatinate, 556 alder samples from 104 sites in Baden-Wuerttemberg and 141 samples from 22 sites in Franconia were examined. Almost all samples (98%) were infected with 16SrV phytoplasmas. The distribution of MAP-FD2 phytoplasmas varied greatly among the different regions: no infections were found in the Mosel-Saar-Ruwer region while along the Rhine valley from Rheinhessen to Baden infection rates around 70% were observed. Northern and Southern regions of Baden-Wuerttemberg as well as Franconia had lower infection rates ranging from 15% to 40%. Insect captures in the different regions showed that O. ishidae is present in all regions but infection rates varied indicating that this invasive species is responsible for the spread of MAP-FD2 phytoplasma in alder in Germany. Only few infections with MAP-FD1 genotypes were found. Specific primers for the epidemic map genotype M54 revealed no infection in the alder trees.

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.

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 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.

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.

Plant-emitted volatile organic compounds play an important role in plant–insect interactions. Thanks to plant-emitted volatiles, herbivores are able to find suitable hosts. Recognition and location of host plants are a key challenge for successful survival and reproduction of migrating insects, such as the plum psyllid Cacopsylla pruni. This psyllid migrates between Prunus spp. for reproduction and conifers for overwintering. C. pruni also is the only known vector of ‘Candidatus Phytoplasma prunorum’, a plant pathogen causing the European Stone Fruit Yellows, a severe plant disease. The preference of C. pruni for different Prunus species was monitored in the field. The sampling revealed a high abundance of C. pruni on Prunus spinosa, the natural host, as well as on different Prunus rootstock suckers. To investigate the influence of volatile profiles from different plants on the host preferences of C. pruni, the volatiles of two reproduction hosts and one overwintering host were sampled and analyzed by gas chromatography and mass spectrometry. The volatile compositions were compared, and important components that lead to the differentiation between plant species and growth stages were identified. Antennal responses of C. pruni females were elicited by eleven plant species and growth stage-specific volatiles, detected by electroantennography. The role of host plant volatiles on the migration behavior and the use of synthetic components in alternative control strategies are discussed.

A survey of genetic diversity of "flavescence dorée" (FD)-related phytoplasmas in grapevines, alders and clematis as well as alder-feeding leafhoppers was conducted in France, Hungary, Germany, Italy and Serbia.Genotyping was based on the housekeeping gene map and on the vmp genes encoding surface variable membrane proteins.Transmission assays of the phytoplasmas were performed with alder and/or grapevine-feeding leafhoppers.The study demonstrated that European alders constitute an original reservoir of FD phytoplasma by hosting a high diversity of FD-related phytoplasma genotypes, also present in non viticultural areas.The alder phytoplasmas grouping in Vmp-I cluster were transmitted by the Macropsinae O. alni, but were not compatible with the FD phytoplasma vector on grapevine the Deltocephalinae Scaphoideus titanus.The alder phytoplasmas in Vmp-II and -III clusters were transmitted by the Deltocephalinae Allygus spp.and Orientus ishidae.Such pre-existing phytoplasmas were compatible with S. titanus transmissibility and can be responsible for the emergence of FD phytoplasma epidemics in grapevine.VmpA proteins of cluster II better adhered to Euscelidius variegatus and S. titanus insect cells and midguts than those of cluster I.Such adhesins might play a key role in the adaptation to new vectors.

Endophytic fungi were isolated from apricot and peach trees which had recovered from European stone fruit yellows disease. To test for their phytoplasma antagonistic activity a plant tissue culture system based on apple proliferation-infected apple plants was used. Six selected fungi were inoculated and their effect on plant growth and phytoplasma concentration was measured. One fungus, Epicoccum nigrum, significantly reduced apple proliferation symptoms and phytoplasma concentration both in in vitro and in ex vitro plants.

Drosophila suzukii (Matsumura) is an invasive species, which is able to attack intact ripening fruit and has become a serious pest in the Americas and Europe. However, susceptibility toward D. suzukii varies strongly within and between grapevine cultivars. The aim of our study was to differentiate between berry parameters influencing oviposition of D. suzukii in grapevine with two complementary approaches. We investigated the influence of berry skin resistance, total soluble solids (a sugar related parameter), acidity and volatile acidity on grape susceptibility; in the first approach at the cultivar level in a field survey, and in the second approach at the single berry level in a laboratory choice experiment. Both approaches revealed that berry skin resistance explained oviposition decidedly better than chemical composition of the berries did: Soft skinned cultivars and berries received significantly more eggs than hard skinned cultivars and berries. These findings suggest a major role of berry skin resistance in the susceptibility of grapevine toward D. suzukii. The cultivar approach identified the cultivars Dornfelder, Trollinger (=Vernatsch, Schiava), Portugieser, Roter Elbling and Cabernet Dorsa to be susceptible, whereas Dakapo, Lemberger (=Blauer Limberger) and Riesling showed no oviposition by D. suzukii. Nevertheless, parameters like previous damage, climate, environment and plant protection may have an additional impact.