A Comparative Analysis of Ash Leaf-Colonizing Bacterial Communities Identifies Putative Antagonists ofHymenoscyphus fraxineus

In the last few years, the alarming spread ofHymenoscyphus fraxineus, the causal agent of ash dieback, has resulted in a substantial threat to native ash stands in central and northern Europe. Since leaves and leaf petioles are the primary infection sites, phyllosphere microorganisms are presumed to interact with the pathogen and are discussed as a source of biocontrol agents. We studied compound leaves from susceptible and visible infection-free trees in four ash stands with a high likelihood of infection to assess a possible variation in the bacterial microbiota, depending on the health status of the trees. The bacterial community was analyzed by culture-independent 16S rRNA gene amplicon sequencing and through the isolation and taxonomic classification of 2,589 isolates using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The bacterial community structure did not show significant differences. However, a set of amplicon sequence variants (ASVs) and MALDI groups belonging toLuteimonas,Aureimonas,Pseudomonas,Bacillus, andPaenibacilluswere distinctly increased in tolerant trees, which may be associated with the ability of the tree to resist the pathogen. The most obvious differences were observed forLuteimonas, a genus that is also exclusively present in the healthy core microbiome. In a firstin vitroscreen of antagonists, approximately 11% of total isolates suppressed the growth ofH. fraxineus, but a statistical test with two differentH. fraxineusstrains confirmed only the antagonistic activity of 8% of these isolates. The antagonistic isolates were assigned toBacillus velezensis,Pantoea vagans, andPseudomonas caspiana.Overall, our study provides a set of isolates or phylogenetic groups that might be involved in the process that prevents the penetration and spread ofH. fraxineus.In the next step,in plantaexperiments are required with a longer period of exposure toH. fraxineusto evaluate effective isolates or consortia of isolates acting through direct antagonism or competition or indirectly by inducing resistance.