Abstract
In the last few years, the alarming spread of Hymenoscyphus 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 to Luteimonas, Aureimonas, Pseudomonas, Bacillus, and Paenibacillus were 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 for Luteimonas, a genus that is also exclusively present in the healthy core microbiome. In a first in vitro screen of antagonists, approximately 11% of total isolates suppressed the growth of H. fraxineus, but a statistical test with two different H. fraxineus strains confirmed only the antagonistic activity of 8% of these isolates. The antagonistic isolates were assigned to Bacillus velezensis, Pantoea vagans, and Pseudomonas 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 of H. fraxineus. In the next step, in planta experiments are required with a longer period of exposure to H. fraxineus to evaluate effective isolates or consortia of isolates acting through direct antagonism or competition or indirectly by inducing resistance.
Highlights
Starting in northeastern Poland in the early 1990s, the severe dieback of common ash (Fraxinus excelsior) with a high mortality rate has spread across the European continent and is present almost throughout the entire natural distribution range of European ash (Kowalski and Holdenrieder, 2009a; McKinney et al, 2014; Vasaitis and Enderle, 2017)
The analysis of the beta diversity resulted in small but significant differences between the bacterial community structure grouped by forest plot and health status (ANOSIM R = 0.2118, P < 0.001)
In ash dieback, which is caused by H. fraxineus, leaflets and petioles are the main entry point for the pathogen (Cleary et al, 2013; Hanackova et al, 2017b)
Summary
Starting in northeastern Poland in the early 1990s, the severe dieback of common ash (Fraxinus excelsior) with a high mortality rate has spread across the European continent and is present almost throughout the entire natural distribution range of European ash (Kowalski and Holdenrieder, 2009a; McKinney et al, 2014; Vasaitis and Enderle, 2017). The causal agent of ash dieback is the invasive ascomycete fungus Hymenoscyphus fraxineus Kowalski) (Baral et al, 2014). This pathogen is considered to originate from Asia, where it is reported to have an asymptotic association with Fraxinus mandshurica and Fraxinus chinensis ssp. In Europe, it is killing ash at an alarming rate and displacing the non-aggressive indigenous fungus Hymenoscyphus albidus (Drenkhan et al, 2017). Less than 5% of trees are partially resistant or tolerant to ash dieback disease, and trees of all ages are affected at various site types in forest, urban, and nursery settings (Timmermann et al, 2011; McKinney et al, 2014). While young trees often die within a few years after infection, older trees generally become chronically diseased and more susceptible to root rot diseases caused by Armillaria spp. (Skovsgaard et al, 2010; Langer et al, 2015; Chandelier et al, 2016)
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