Michael Maixner

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

ABSTRACT 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.

Invasion biology and host specificity of the grapevine yellows disease vector Hyalesthes obsoletus in Europe

Within the past 10 years, the yellows disease ‘bois noir’ (BN) has become one of the commercially most important diseases of grapevine [Vitis vinifera L. (Vitaceae)] in Europe. Infection pressure is caused by phytoplasmas of the stolbur 16SrXII‐A group that are transmitted by a planthopper vector, Hyalesthes obsoletus Signoret (Homoptera: Auchenorrhyncha). Infestation happens as an accidental side‐effect of the feeding behaviour of the vector, as vector and pathogen proliferation is dependent on other plants. In Germany, the increase of BN is correlated with the use of a new host plant by the vector, increase in abundance of the vector on the new host plant, and dissemination of host plant‐specific pathogen strains. In this article, we investigate geographic and host‐associated range expansion of the vector. We test whether host‐plant utilization in Germany, hence the increase in BN, is related to genetic host races of the vector and, if so, whether these have evolved locally or have immigrated from southern populations that traditionally use the new host plant. The genetic population analysis demonstrates a recent expansion and circum‐alpine invasion of H. obsoletus into German and northern French wine‐growing regions, which coincides with the emergence of BN. No H. obsoletus mitochondrial DNA haplotype host‐plant affiliation was found, implying that the ability to use alternative host plants is genetically intrinsic to H. obsoletus. However, subtle yet significant random amplified polymorphic DNA (RAPD) genetic differentiation was found among host plant populations. When combined, these results suggest that a geographic range expansion of H. obsoletus only partly explains the increase of BN, and that interactions with host plants also occur. Further possible beneficial factors to H. obsoletus, such as temperature increase and phytoplasma interactions, are discussed.

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 vectors 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 pathogens 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 vectors historical area of origin. The introduction of tuf-a to stinging nettle in the northern range was therefore independent of vectors host-plant specialisation but the rapid pathogen dissemination depended on the vectors host shift, whereas the general dissemination elsewhere was linked to plant specialisation of the pathogen but not of the vector.

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 vectors host range may increase transmission potential, whereas specialization may induce specific disease cycles. The process leading to a vectors 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 < FHT < 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.

Infection of Bois-Noir tuf-type-I stolbur phytoplasma in Hyalesthes obsoletus (Hemiptera: Cixiidae) larvae and influence on larval size.

Recent dramatic spread of the grapevine yellows disease Bois Noir (BN) in Germany is above all explained by highly increased abundances of the vector Hyalesthes obsoletus (Hemiptera: Cixiidae) associated to the plant Urtica dioica, the reservoir of the BN pathogen stolbur tuf‐type‐I. The vector acquires BN‐phytoplasma as larvae whilst feeding on the roots of infected U. dioica. To understand the dynamics of the Urtica‐cycle, we tested at what instar larvae become infected and whether infection affects larvae size (i.e. growth) at two sites in the Mosel Valley, Germany. Larvae were tested from infected plants and collected at instar‐stages 3, 4 and 5. Larvae at stage 3 were already infected but infection rates increased significantly between stage 3 and 5, mean infection rates: 0.12–0.62. There was no effect of infection on larval size at any instar stage.

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 vectors host range and the pathogens reservoir range. Because vectors interact with pathogens, the direction and strength of a vectors 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.

Leafhoppers and Planthoppers: Their Bionomics, Pathogen Transmission and Management in Vineyards

Auchenorrhyncha is the hemipteran suborder that includes cicadas, leafhoppers, froghoppers or spittlebugs, planthoppers and treehoppers. Leafhoppers (Cicadellidae) are cosmopolitan and one of the largest insect families with approximately 22,000 described species (Forero 2008). Planthoppers (infra-order Fulgoromorpha) are mainly tropical with approximately 20 described families (Urban and Cryan 2007). Leafhoppers and planthoppers have piercing-sucking mouthparts that cause direct damage to plants by feeding in mesophyll cells or on xylem and/or phloem sap, and indirect damage by transmitting pathogens. Because pathogens are not easily managed in plants, the most common control methods rely on the use of insecticides to manage insect populations. This chapter will provide an overview of the life cycles, feeding behavior and vector abilities of leafhoppers and planthoppers causing damage in vineyards. Present and future management methods will be presented in a viticultural context.

Application of next-generation sequencing for simultaneousdetection of viruses, viroids and phytoplasmas in grapevine and fruit trees

Recent studies on endophytic Kosakonia radicincitans DSM 16656T demonstrated a wide plant growth-promoting activity. The application of this Gram-negative bacterium as a biofertilizer is limited due to the lack of adequate formulation that protects the cells during drying and storage and supports plant colonization. Here we set out to elucidate the compatible solutes accumulation influence of K. radicincitans triggered by osmotic stress on its capacity as an endophyte in radish plants. We found that physiological modifications by osmotic stress treatments and accumulation of compatible solutes during cultivation, improve the capability of K. radicincitans formulated in dry beads to colonize and to promote radish growth. Thus, pre-conditioning of cells with NaCl 4% and by adding compatible solutes such as hydroxyectoine during cultivation induced a positive effect on relative gene expression response, enhancing significantly the ability to colonize plant tissue up to 10 fold. Additionally, when this osmolyte was added, either dry matter of tuber or leaves increased by 3 % and 13.59 %, respectively in comparison to beads without the bacterium. These first results indicate that a systematic approach to cultivation and formulation may increase the endophytic capacity of this bacterium.

XF-ACTORS – Ein internationales Forschungsprojekt zur Vermeidung der Einschleppung und Ausbreitung von Xylella fastidiosa

Recent studies on endophytic Kosakonia radicincitans DSM 16656T demonstrated a wide plant growth-promoting activity. The application of this Gram-negative bacterium as a biofertilizer is limited due to the lack of adequate formulation that protects the cells during drying and storage and supports plant colonization. Here we set out to elucidate the compatible solutes accumulation influence of K. radicincitans triggered by osmotic stress on its capacity as an endophyte in radish plants. We found that physiological modifications by osmotic stress treatments and accumulation of compatible solutes during cultivation, improve the capability of K. radicincitans formulated in dry beads to colonize and to promote radish growth. Thus, pre-conditioning of cells with NaCl 4% and by adding compatible solutes such as hydroxyectoine during cultivation induced a positive effect on relative gene expression response, enhancing significantly the ability to colonize plant tissue up to 10 fold. Additionally, when this osmolyte was added, either dry matter of tuber or leaves increased by 3 % and 13.59 %, respectively in comparison to beads without the bacterium. These first results indicate that a systematic approach to cultivation and formulation may increase the endophytic capacity of this bacterium.

Development of methods for pre-symptomatic detection of grapevine diseases like esca, phytoplasmoses and viruses using hyperspectral sensors

Ryegrass (Lolium spec.) is the most important cool-season forage crop in temperate regions. Though, the seed production is considerably affected by several fungal and bacterial obligate biotrophic pathogens. The overall purpose of this study is directed to developing ryegrass cultivars with multiple pathogen resistance and agronomic adaption to Germany’s agricultural conditions. This aim shall be achieved by combining genes for resistances to stem rust, crown rust and bacterial wilt. The pyramidisation shall be accomplished by the use of specific molecular markers which will be derived by bulked segregant analysis combined with next generation sequencing based massive analysis of cDNA ends (MACE) transcriptome profiling. RNA was isolated from bulks of infected and noninfected leaf segments from susceptible and resistant genotypes of various fullsibling mapping populations (n ≥ 200) and their respective parental lines for every investigated pathogen. After MACE was performed, bioinformatic analysis detects SNPs and transcripts that were exclusively expressed in the resistant bulk. Thus, 30 molecular markers were genetically mapped to a 50.8 cM spanning region surrounding the stem rust resistance locus LpPg1. The development of this high efficient molecular selection tool marks MACE as a fast and reliable method that detects polymorphisms for genetic mapping of candidate genes and obtains to be the method of choice for investigating the molecular and genetic base of resistances to stem rust, crown rust and bacterial wilt.The current production systems in arable farming have reached their limits. Sizes of machinery are continuously increasing. Compaction and limits on the road are the consequences. Productionrelated restrictions like nitrogen pressure and development of resistances against plant protection products are further problems. Last but not least, the sociopolitical acceptance of crop production is questioned in public opinion. Due to these circumstances the question arises if the system of crop farming which has been adapted to the machinery available on the market is the right strategy for the future. Why not going the other way round and decide what a plant production system has to look like to be at an optimum and then decide what kind of machinery is needed to cultivate? Following this idea the plants must be in the focus.