Andreas Ulrich

Background: Forests cover large areas of the terrestrial land surface and play a key role in climate regulation and biodiversity conservation. Tree-associated microbial communities are integral components of forest ecosystems and contribute to plant functioning, stress tolerance, and interactions with the environment. However, the factors shaping the phyllosphere microbiome of forest trees across large environmental gradients remain less explored. Here, we investigated the bacterial leaf microbiome of Fraxinus excelsior across different climatic regions in Germany to identify its core microbiome and determine the relative importance of environmental and host-related factors shaping the phyllosphere microbiome of forest trees across large environmental gradients assembly. Therefore, 30 ash trees were sampled at each of the 12 sites across Germany, at which we assessed their leaf microbiome, leaf physio-chemical properties and a set of site parameters. Results: In total, 755 genera were detected. Only three genera Hymenobacter , Massilia and Sphingomonas were predominant across all sites. Geographical position, precipitation, tree age and the leaf and soil chemistry parameters were significant factors that explained 22.1% of variance between microbiome structures between the different sites. Seven genera were identified as the core leave microbiome of F. excelsior . The core microbiome assessed for each sampling site was mostly more diverse and differed between sites. Complex co-occurrence networks separated by sites with the identified six hub bacterial taxa proved that they were essential for the local core microbiomes. However, the network complexity differed significantly between the sampling sites. Conclusion: Our findings clearly indicate the existence of an ash core microbiome on leaves that occurred at all sites but was locally differently interconnected to further microbiome members. The network analyses revealed that less frequent genera play important roles as hub taxa. Furthermore, we were also able to prove the modulating role of biotic and abiotic factors on the ash phyllosphere microbiome. An optimized core microbiome may be considered crucial for improving tolerance against plant diseases, such as ash dieback, in future.

Since ash dieback has been recognized as a major threat to Fraxinus excelsior, multiple lines of research have focused on potential management to mitigate this disease. One area of focus has been the microbiome of the trees and the application of biocontrol agents (BCAs). In this study, we aimed to find suitable BCAs that can help control Hymenoscyphus fraxineus and the resulting ash dieback. Therefore, we studied the health of ash seedlings under nursery conditions via inoculation with beneficial bacterial isolates and consortia to suppress the pathogen. By using offspring of two tolerant ash trees, we aimed to optimize this via synergistic effects. We found that Schauerella fraxinea B3P038 and Luteimonas fraxinea D4P002 lowered the symptoms of ash dieback. Using strain-specific qPCR assays, only the persistence of Aureimonas altamirensis C2P003 could be proven in the inoculated ash seedlings. Similarly, the fungal pathogen was also unable to infect the plants during the summer months. In addition, we were able to show that the inoculated bacterial strains influenced the microbiome, even without persisting on the plants. These results are encouraging, as positive effects can be taken from the greenhouse to the field and further studies should follow up on the B3P038, C2P003 and D4P002 strains. However, under field conditions, a strong variation in the effects must be considered. This strongly suggests that in addition to plant scoring, monitoring inoculation success as well as pathogen abundance is necessary.

Some European ash trees show tolerance towards dieback caused by the invasive pathogen Hymenoscyphus fraxineus. The microbiome of these trees harbours a range of specific bacterial groups. One of these groups belonging to the species Aureimonas altamirensis was studied in detail by genome analysis and a plant inoculation trial. The strain group was shown to be phylogenetically distinct from clinical isolates by 16S rRNA analysis and phylogenomics. Genome analysis of a representative strain C2P003 resulted in a large number of unique gene sequences in comparison to other well-studied strains of the species. A functional analysis of the genome revealed features associated with the synthesis of exopolysaccharides, protein secretion and biofilm production as well as genes for stress adaptation, suggesting the ability of C2P003 to effectively colonize ash leaves. The inoculation of ash seedlings with C2P003 showed a significant positive effect on the plant health of the seedlings that were exposed to H. fraxineus infection. This effect was maintained over a period of three years and was accompanied by a significant shift in the bacterial microbiome composition one year after inoculation. Overall, the results indicate that C2P003 may suppress H. fraxineus in or on ash leaves via colonization resistance or indirectly by affecting the microbiome.

Microorganisms acting as sinks for the greenhouse gas nitrous oxide (N₂O) are gaining increasing attention in the development of strategies to control N₂O emissions. Non-denitrifying N₂O reducers are of particular interest because they can provide a real sink without contributing to N₂O release. The bacterial strain under investigation (IGB 4-14^T), isolated in a mesocosm experiment to study the litter decomposition of Phragmites australis (Cav.), is such an organism. It carries only a nos gene cluster with the sec-dependent Clade II nosZ and is able to consume significant amounts of N₂O under anoxic conditions. However, consumption activity is considerably affected by the O₂ level. The reduction of N₂O was not associated with cell growth, suggesting that no energy is conserved by anaerobic respiration. Therefore, the N₂O consumption of strain IGB 4-14^T rather serves as an electron sink for metabolism to sustain viability during transient anoxia and/or to detoxify high N₂O concentrations. Phylogenetic analysis of 16S rRNA gene similarity revealed that the strain belongs to the genus Flavobacterium. It shares a high similarity in the nos gene cluster composition and the amino acid similarity of the nosZ gene with various type strains of the genus. However, phylogenomic analysis and comparison of overall genome relatedness indices clearly demonstrated a novel species status of strain IGB 4-14^T, with Flavobacterium lacus being the most closely related species. Various phenotypic differences supported a demarcation from this species. Based on these results, we proposed a novel species Flavobacterium azooxidireducens sp. nov. (type strain IGB 4-14^T = LMG 29709^T = DSM 103580^T).

Three strains (H4-D09^T, S2-D11 and S9-F39) of a member of the genus Paracoccus attributed to a novel species were isolated from topsoil of temperate grasslands. The genome sequence of the type strain H4-D09^T exhibited a complete set of genes required for denitrification as well as methylotrophy. The genome of H4-D09^T included genes for two alternative pathways of formaldehyde oxidation. Besides the genes for the canonical glutathione (GSH)-dependent formaldehyde oxidation pathway, all genes for the tetrahydrofolate–formaldehyde oxidation pathway were identified. The strain has the potential to utilize methanol and/or methylamine as a single carbon source as evidenced by the presence of methanol dehydrogenase (mxaFI) and methylamine dehydrogenase (mau) genes. Apart from dissimilatory denitrification genes (narA, nirS, norBC and nosZ), genes for assimilatory nitrate (nasA) and nitrite reductases (nirBD) were also identified. The results of phylogenetic analysis based on 16S rRNA genes coupled with riboprinting revealed that all three strains represented the same species of genus Paracoccus . Core genome phylogeny of the type strain H4-D09^T indicated that Paracoccus thiocyanatus and Paracoccus denitrificans are the closest phylogenetic neighbours. The average nucleotide index (ANI) and digital DNA–DNA hybridization (dDDH) with the closest phylogenetic neighbours revealed genetic differences at the species level, which were further substantiated by differences in several physiological characteristics. The major respiratory quinone is Q-10, and the predominant cellular fatty acids are C_18 : 1ω7c, C_19 : 0cyclo ω7c, and C_16 : 0, which correspond to those detected in other members of the genus. The polar lipid profile consists of a diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylcholine (PC), aminolipid (AL), glycolipid (GL) and an unidentified lipid (L). On the basis of our results, we concluded that the investigated isolates represent a novel species of the genus Paracoccus , for which the name Paracoccus methylovorus sp. nov. (type strain H4-D09^T=LMG 31941^T= DSM 111585^T) is proposed.

Plant-associated Stenotrophomonas isolates have great potential for plant growth promotion, especially under stress conditions, due to their ability to promote tolerance to abiotic stresses such as salinity or drought. The endophytic strain Stenotrophomonas sp. 169, isolated from a field-grown poplar, increased the growth of inoculated in vitro plants, with a particular effect on root development, and was able to stimulate the rooting of poplar cuttings in the greenhouse. The strain produced high amounts of the plant growth-stimulating hormone auxin under in vitro conditions. The comparison of the 16S rRNA gene sequences and the phylogenetic analysis of the core genomes showed a close relationship to Stenotrophomonas chelatiphaga and a clear separation from Stenotrophomonas maltophilia. Whole genome sequence analysis revealed functional genes potentially associated with attachment and plant colonization, growth promotion, and stress protection. In detail, an extensive set of genes for twitching motility, chemotaxis, flagella biosynthesis, and the ability to form biofilms, which are connected with host plant colonization, could be identified in the genome of strain 169. The production of indole-3-acetic acid and the presence of genes for auxin biosynthesis pathways and the spermidine pathway could explain the ability to promote plant growth. Furthermore, the genome contained genes encoding for features related to the production of different osmoprotective molecules and enzymes mediating the regulation of stress tolerance and the ability of bacteria to quickly adapt to changing environments. Overall, the results of physiological tests and genome analysis demonstrated the capability of endophytic strain 169 to promote plant growth. In contrast to related species, strain 169 can be considered non-pathogenic and suitable for biotechnology applications.

A novel strain was isolated from grassland soil that has the potential to assimilate ammonium by the reduction of nitrate in the presence of oxygen. Whole genome sequence analysis revealed the presence of an assimilatory cytoplasmic nitrate reductase gene nasA and the assimilatory nitrite reductase genes nirBD which are involved in the sequential reduction of nitrate to nitrite and further to ammonium, respectively. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the isolate represents a member of the genus Pseudomonas . The closest phylogenetic neighbours based on 16S rRNA gene sequence analysis are the type strains of Pseudomonas peli (98.17%) and Pseudomonas guineae (98.03%). In contrast, phylogenomic analysis revealed a close relationship to Pseudomonas alcaligenes . Computation of the average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) with the closest phylogenetic neighbours of S1-A32-2^T revealed genetic differences at the species level, which were further substantiated by differences in several physiological characteristics. On the basis of these results, it was concluded that the soil isolate represents a novel species of the genus Pseudomonas, for which the name Pseudomonas campi sp. nov. (type strain S1-A32-2^T=LMG 31521^T=DSM 110222^T) is proposed.