Fungal and bacterial communities and their associations in snow-free and snow covered (sub-)alpine Pinus cembra forest soils.

BACKGROUND: In Europe, Pinus cembra forests cover subalpine and alpine areas and they are of high conservational and ecological relevance. These forests experience strong seasonality with alternating snow-free and snow covered periods. Although P. cembra is known for mycorrhization and mycorrhizae usually involve fungi, plants and bacteria, the community compositions of fungi and bacteria and their associations in (sub-)alpine P. cembra forests remain vastly understudied. Here, we studied the fungal and bacterial community compositions in three independent (sub-)alpine P. cembra forests and inferred their microbial associations using marker gene sequencing and network analysis. We asked about the effect of snow cover on microbial compositions and associations. In addition, we propose inferring microbial associations across a range of filtering criteria, based on which we infer well justified, concrete microbial associations with high potential for ecological relevance that are typical for P. cembra forests and depending on snow cover. RESULTS: The overall fungal and bacterial community structure was comparable with regards to both forest locations and snow cover. However, occurrence, abundance, and diversity patterns of several microbial taxa typical for P. cembra forests differed among snow-free and snow covered soils, e.g. Russula, Tetracladium and Phenoliphera. Moreover, network properties and microbial associations were influenced by snow cover. Here, we present concrete microbial associations on genus and species level that were repeatedly found across microbial networks, thereby confirming their ecological relevance. Most importantly, ectomycorrhizal fungi, such as Basidioascus, Pseudotomentella and Rhizopogon, as well as saprobic Mortierella changed their bacterial association partners depending on snow cover. CONCLUSION: This is the first study researching fungal-bacterial associations across several (sub-)alpine P. cembra forests. The poorly investigated influence of snow cover on soil fungi and bacteria, especially those mycorrhizing P. cembra roots, but also saprobic soil organisms, underlines the relevance of forest seasonality. Our findings highlight that the seasonal impact of snow cover has significant consequences for the ecology of the ecosystem, particularly in relation to mycorrhization and nutrient cycling. It is imperative to consider such effects for a comprehensive understanding of the functioning resilience and responsiveness of an ecosystem.

Co-cultivation of Mortierellaceae with Pseudomonas helmanticensis affects both their growth and volatilome.

Volatile organic compounds (VOCs) might mediate microbial interactions, especially in spatially structured environments, such as soil. However, the variety and specificity of VOC production are poorly understood. Here, we studied 25 Mortierellaceae strains belonging to the genera Linnemannia and Entomortierella in both pure and co-culture with Pseudomonas helmanticensis under laboratory conditions. We analysed both the fungal growth depending on co-cultivation and the cultures' volatilomes applying proton-transfer-reaction time-of-flight and gas chromatography-mass spectrometry (PTR-ToF-MS and GC-MS). In a strain-specific manner, we found the fungi's radial growth rate and colony morphology affected by the presence of P. helmanticensis. The fungus seemed to generally reduce the bacterial growth. The volatilomes of the fungal and bacterial pure and co-cultures were diverse. While the fungi frequently consumed VOCs, P. helmanticensis produced a higher diversity and amount of VOCs than any fungal strain. Our results support that both the pure and co-culture volatilomes are taxonomically conserved. Taken together, our data supports the relevance of VOCs in Mortierellaceae-P. helmanticensis interaction. We also discuss individual VOCs that appear relevant in the interaction.

Habitat, Snow-Cover and Soil pH, Affect the Distribution and Diversity of Mortierellaceae Species and Their Associations to Bacteria.

Mortierellaceae species are among the most frequent and globally distributed soil fungi. However, the factors shaping their diversity and distribution remain obscure. Several species have been reported to be associated to bacteria, but the kind and frequency of such associations were not addressed up to now. We hypothesized that such associations could be important for Mortierellaceae ecology. Therefore, our aim was to understand the driving factors responsible for the Mortierellaceae diversity, community composition and bacterial associations in alpine and subalpine habitats. For answering our question, we collected both snow-free and snow-covered soil at sampling sites from different habitats: bare alpine soil in a glacier forefield, alpine dwarf-willow habitats, and high-altitude Pinus cembra forests. The isolations were carried out by direct cultivation without any antibiotics to the isolation media. Altogether, we obtained 389 Mortierellaceae isolates representing 29 operational taxonomic units (OTUs). Many OTUs could be placed to the genera Mortierella sensu stricto, Dissophora, Entomortierella, Gamsiella, Linnemannia, and Podila, but others could not unambiguously be assigned to a genus. Our results demonstrate that both, the distribution as well as the diversity of the Mortierellaceae species, were significantly influenced by habitat, soil pH, and snow-cover. We noticed that >30% of our isolates were associated to a non-contaminant bacterium. The bacteria associated to our Mortierellaceae isolates belonged to seven different genera. Pseudomonas was the most frequently detected genus associated to the isolated Mortierellaceae species and it was found to be species-specific. Mortierellaceae-bacteria pairs, including those with Pseudomonas, were influenced by location, habitat, and snow-cover. The majority of the fungus-bacterium associations were potentially epihyphal, but we also detected potential endohyphal bacterial species belonging to Mycoavidus, Burkholderiaceae, and Paraburkholderia. Taken together, the non-random associations we detected suggest that fungus-bacterium associations are ecologically meaningful - an interesting path that needs to be investigated further.

The impact of episporic modification of Lichtheimia corymbifera on virulence and interaction with phagocytes.

Fungal infections caused by the ancient lineage Mucorales are emerging and increasingly reported in humans. Comprehensive surveys on promising attributes from a multitude of possible virulence factors are limited and so far, focused on Mucor and Rhizopus. This study addresses a systematic approach to monitor phagocytosis after physical and enzymatic modification of the outer spore wall of Lichtheimia corymbifera, one of the major causative agents of mucormycosis. Episporic modifications were performed and their consequences on phagocytosis, intracellular survival and virulence by murine alveolar macrophages and in an invertebrate infection model were elucidated. While depletion of lipids did not affect the phagocytosis of both strains, delipidation led to attenuation of LCA strain but appears to be dispensable for infection with LCV strain in the settings used in this study. Combined glucano-proteolytic treatment was necessary to achieve a significant decrease of virulence of the LCV strain in Galleria mellonella during maintenance of the full potential for spore germination as shown by a novel automated germination assay. Proteolytic and glucanolytic treatments largely increased phagocytosis compared to alive resting and swollen spores. Whilst resting spores barely (1-2%) fuse to lysosomes after invagination in to phagosomes, spore trypsinization led to a 10-fold increase of phagolysosomal fusion as measured by intracellular acidification. This is the first report of a polyphasic measurement of the consequences of episporic modification of a mucormycotic pathogen in spore germination, spore surface ultrastructure, phagocytosis, stimulation of Toll-like receptors (TLRs), phagolysosomal fusion and intracellular acidification, apoptosis, generation of reactive oxygen species (ROS) and virulence.

High-Throughput Volatilome Fingerprint Using PTR-ToF-MS Shows Species-Specific Patterns in Mortierella and Closely Related Genera.

In ecology, Volatile Organic Compounds (VOCs) have a high bioactive and signaling potential. VOCs are not only metabolic products, but are also relevant in microbial cross talk and plant interaction. Here, we report the first large-scale VOC study of 13 different species of Mortierella sensu lato (s. l.) isolated from a range of different alpine environments. Proton Transfer Reaction-Time-of-Flight Mass Spectrometry (PTR-ToF-MS) was applied for a rapid, high-throughput and non-invasive VOC fingerprinting of 72 Mortierella s. l. isolates growing under standardized conditions. Overall, we detected 139 mass peaks in the headspaces of all 13 Mortierella s. l. species studied here. Thus, Mortierella s. l. species generally produce a high number of different VOCs. Mortierella species could clearly be discriminated based on their volatilomes, even if only high-concentration mass peaks were considered. The volatilomes were partially phylogenetically conserved. There were no VOCs produced by only one species, but the relative concentrations of VOCs differed between species. From a univariate perspective, we detected mass peaks with distinctively high concentrations in single species. Here, we provide initial evidence that VOCs may provide a competitive advantage and modulate Mortierella s. l. species distribution on a global scale.