The ash dieback pathogen Hymenoscyphus fraxineus threatens European ash (Fraxinus excelsior) since its introduction into Europe in the early 1990s. European-wide population genetics analysis has been previously carried out and revealed that the fungus population had weak structure and was a sexually reproducing species. To date, no work has been done to understand the population dynamics at a more local scale, and in particular along a tree species diversity gradient. In this study, the population structure of H. fraxineus was investigated in plots of varying tree species mixture containing F. excelsior in Hainich National Park, Germany. Fallen previous years' ash petioles were collected in July 2012 from targeted ash trees. DNA was extracted and microsatellite markers were amplified. We aim to determine the population genetics structure and understand the infection biology of the pathogen.

In addition to the collection fresh leaves within task 4.6, we collected fallen ash petioles to perform molecular analysis on population genetic structure of the specific pathogen Hymenoscyphus fraxineus from Fraxinus excelsior. Trees from three plots that were previously selected for leaf sampling, were selected for petiole sampling. There were four trees from the monoculture plot (GER04), three trees from the 2 species mixture (GER03), and three trees from the 4 species mixture (GER37), and two trees from the 4 species mixture (GER31). Approximately 45 petioles were collected within 1 m around each stem of targeted trees.

Exploratory Region: Germany

Between July 9, 2012 and July 16, 2012

Fallen previous year's ash petioles collected within 1 m around the stem of targeted Fraxinus excelsior

Additional petiole samples were collected from other ash trees within the plots with Fraxinus excelsior, but were not processed in the above described methods as a result of cost constraints. Thus data for these are not presented here.

Each petiole was separated into petiole sections at each leaf node, and each sample is a one such petiole section. After confirmation of the presence of the fungus within ash petiole sections with species-specific primers, 14 previously published microsatellite markers will be amplified to determine the genotype of the fungus within each petiole section. Genotypic diversity will be calculated from the given fragment lengths. Principal Coordinates Analysis will be conducted to determine the spatial distribution of the genotypes. Analysis of molecular variance will determine the degree of genetic differentiation. Structure analysis will aid in the investigation of the existence of population structure. The index of association will be calculated to determine the reproductive mode of the fungus.