Analyzing Ash Leaf-Colonizing Fungal Communities for Their Biological Control of .

The invasive ascomycete has been threatening populations throughout Europe for over two decades. Since the infection and first colonization by the pathogen occurs in leaves, leaf-colonizing microorganisms have been discussed as a barrier and as possible biocontrol agents against the disease. To identify fungal groups with health-supporting potential, we compared the fungal microbiota of compound leaves from susceptible and tolerant ash trees in four ash stands with high exposure. The fungal communities were analyzed both culture-independently by ITS2 amplicon sequencing and by the taxonomic classification of 1,704 isolates using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) or sequencing of the entire ITS region. The fungal community structure did not show significant differences depending on the health status. However, for several OTUs and a MALDI group, a significantly higher abundance was found in tolerant ash trees. Thus, the yeast was significantly increased and accounted for 12.3% of the mycobiome of tolerant ashes (OTU0003), and it had also a distinctly higher abundance among the isolates. The filamentous ascomycete was increased 24-fold among the isolates of tolerant trees, but its abundance was comparably low. An screening for the growth inhibition of the pathogen cocultivation resulted in 28 yeast-like isolates and 79 filamentous fungi with antagonistic activity. A statistical cocultivation test on two strains confirmed six of the yeast-like isolates that suppressed significantly, from 39-50%, two of them through a fungicidal effect. The highest inhibition rates among the yeasts were found for three isolates belonging to and . The cocultivation test of the filamentous isolates revealed higher effects compared to the yeasts. Four isolates showed significant inhibition of both strains with a rate of 72-100%, and five further isolates inhibited only one strain significantly. The most effective isolates were members of the genus . During the next step, tests will be necessary to verify the efficacy of the antagonistic isolates and to assess their suitability as biocontrol agents.