Fraxinus excelsior populations are in decline due to the ash dieback disease Hymenoscyphus fraxineus. It is important to understand genotypic and environmental effects on its fungal microbiome to develop disease management strategies. To do this, we used culture dependent and culture independent approaches to characterize endophyte material from contrasting ash provenances, environments, and tissues (leaves, roots, seeds). Endophytes were isolated and identified using nrITS, LSU, or tef DNA loci in the culture dependent assessments, which were mostly Ascomycota and assigned to 37 families. Few taxa were shared between roots and leaves. The culture independent approach used high throughput sequencing (HTS) of nrITS amplicons directly from plant DNA and detected 35 families. Large differences were found in OTU diversity and community composition estimated by the contrasting approaches and these data need to be combined for estimations of the core endophyte communities. Species richness and Shannon index values were highest for the leaf material and the French population. Few species were shared between seed and leaf tissue. PCoA and NMDS of the HTS data showed that seed and leaf microbiome communities were highly distinct and that there was a strong influence of Fraxinus species identity on their fungal community composition. The results will facilitate a better understanding of ash fungal ecology and are a step toward identifying microbial biocontrol systems to minimize the impact of the disease.
Keywords: DNA; Fraxinus excelsior; ash; barcoding; communities; core microbiome; dieback; endophyte; genotype; next generation sequencing.