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Abstract

Flavescence dorée (FD) phytoplasma and related 16SrV phytoplasmas are associated with grapevine disease, hazelnut decline, and non-cultivated plants. Their epidemiology is commonly investigated using gene-based typing, especially genotyping of the map gene, which enables rapid comparison of closely related strains. However, identical map genotypes in different hosts do not prove genome-level relatedness. In this thesis, we examined the distribution of 16SrV phytoplasmas in grapevine, hazelnut, and two leafhoppers (Scaphoideus titanus and Orientus ishidae). We evaluated whether selected cross-host relationships inferred from map genotyping are supported by genome-wide data.
The first objective was to use map genotyping and additional genetic markers to compare 16SrV phytoplasmas from grapevine, hazelnut, and leafhoppers in Slovenia. It revealed a partial overlap between grapevine and hazelnut. Some map genotypes previously identified in grapevine outbreaks were also found in hazelnut, and the same genotypes were present in O. ishidae collected from infected hazelnut orchards. First reports from the Czech Republic and the Netherlands generated in this work provided additional European context: FD-related phytoplasmas were detected in grapevine and Clematis vitalba, while a non-epidemic 16SrV genotype was found in asymptomatic alder. These findings suggest that hazelnut and non-cultivated plants in the studied landscapes contribute to the ecological background in which FD-related 16SrV phytoplasmas persist. However, only part of this diversity is directly relevant to grapevine epidemics.
To extend single-locus genotyping towards genome-wide comparison, the second objective addressed sequencing directly from naturally infected plant material. Because phytoplasmas cannot be cultured in cell-free media and occur at low titre in plant tissues, field-sample sequencing is constrained by host DNA background. We developed an enrichment protocol using naturally infected grapevine material. The optimised approach increased phytoplasma read recovery and enabled reconstruction of a high-quality draft genome without propagation in test plants. This enabled the transition from marker-based comparison to genome-wide analysis.
The third objective applied genome-wide analyses to FD-related 16SrV phytoplasmas from grapevine, hazelnut, S. titanus, and O. ishidae. Genome-wide similarity patterns were consistent with map genotype classification for M38 and M50, indicating that map-based classification captured major genomic relationships in this dataset. Host species did not correspond to genome-wide differentiation, while variation within map genotype-related groups indicated an influence of geographic origin. Genome-scale analysis, therefore, added resolution beyond map typing by showing that, for the studied map genotypes, shared genotypes across hosts were also closely related at the genome level, while finer variation was linked more strongly to sampling area than to host.
The thesis supports the interpretation that, for the analysed map genotypes and sampled regions, FD-related 16SrV phytoplasmas occur in a shared multi-host system in which hazelnut is part of the epidemiological landscape rather than an isolated host. It provides a method for directly sequencing phytoplasmas from field material, enabling future comparative genomic studies without prior propagation in test plants.