Legume rhizodeposition promotes nitrogen fixation by soil microbiota under crop diversification.

Biological nitrogen fixation by free-living bacteria and rhizobial symbiosis with legumes plays a key role in sustainable crop production. Here, we study how different crop combinations influence the interaction between peanut plants and their rhizosphere microbiota via metabolite deposition and functional responses of free-living and symbiotic nitrogen-fixing bacteria. Based on a long-term (8 year) diversified cropping field experiment, we find that peanut co-cultured with maize and oilseed rape lead to specific changes in peanut rhizosphere metabolite profiles and bacterial functions and nodulation. Flavonoids and coumarins accumulate due to the activation of phenylpropanoid biosynthesis pathways in peanuts. These changes enhance the growth and nitrogen fixation activity of free-living bacterial isolates, and root nodulation by symbiotic Bradyrhizobium isolates. Peanut plant root metabolites interact with Bradyrhizobium isolates contributing to initiate nodulation. Our findings demonstrate that tailored intercropping could be used to improve soil nitrogen availability through changes in the rhizosphere microbiome and its functions.

A slow-fast trait continuum at the whole community level in relation to land-use intensification.

Organismal functional strategies form a continuum from slow- to fast-growing organisms, in response to common drivers such as resource availability and disturbance. However, whether there is synchronisation of these strategies at the entire community level is unclear. Here, we combine trait data for >2800 above- and belowground taxa from 14 trophic guilds spanning a disturbance and resource availability gradient in German grasslands. The results indicate that most guilds consistently respond to these drivers through both direct and trophically mediated effects, resulting in a 'slow-fast' axis at the level of the entire community. Using 15 indicators of carbon and nutrient fluxes, biomass production and decomposition, we also show that fast trait communities are associated with faster rates of ecosystem functioning. These findings demonstrate that 'slow' and 'fast' strategies can be manifested at the level of whole communities, opening new avenues of ecosystem-level functional classification.

Invasive earthworms shift soil microbial community structure in northern North American forest ecosystems.

Invasive earthworms colonize ecosystems around the globe. Compared to other species' invasions, earthworm invasions have received little attention. Previous studies indicated their tremendous effects on resident soil biota representing a major part of the terrestrial biodiversity. We investigated effects of earthworm invasion on soil microbial communities in three forests in North America by conducting DNA sequencing of soil bacteria, fungi, and protists in two soil depths. Our study shows that microbial diversity was lower in highly invaded forest areas. While bacterial diversity was strongly affected compared to fungi and protists, fungal community composition and family dominance were strongly affected compared to bacteria and protists. We found most species specialized on invasion in fungi, mainly represented by saprotrophs. Comparably, few protist species, mostly bacterivorous, were specialized on invasion. As one of the first observational studies, we investigated earthworm invasion on three kingdoms showing distinct taxa- and trophic level-specific responses to earthworm invasion.

It's time to consider the Arcellinida shell as a weapon.

The shells of testate amoebae are morphologically diverse and persistent in the environment. Accordingly, the examination of the morphology and composition of shells became a standard tool in ecological, palaeoecological, and evolutionary studies. However, so far the function of the shell remains poorly understood and, although based on limited evidence, the shell was considered as a defense mechanism. Based on recent evidence, we propose that the shell of arcellinid testate amoebae is a crucial component facilitating the amoebae's attack of large prey. Accordingly, the shell is not purely protective, but must be considered also as a weapon. This change in perspective opens up numerous new avenues in protistology and will lead to a substantial change in ecological, palaeoecological, and evolutionary research.

Protistan predation selects for antibiotic resistance in soil bacterial communities.

Understanding how antibiotic resistance emerges and evolves in natural habitats is critical for predicting and mitigating antibiotic resistance in the context of global change. Bacteria have evolved antibiotic production as a strategy to fight competitors, predators and other stressors, but how predation pressure of their most important consumers (i.e., protists) affects soil antibiotic resistance genes (ARGs) profiles is still poorly understood. To address this gap, we investigated responses of soil resistome to varying levels of protistan predation by inoculating low, medium and high concentrations of indigenous soil protist suspensions in soil microcosms. We found that an increase in protistan predation pressure was strongly associated with higher abundance and diversity of soil ARGs. High protist concentrations significantly enhanced the abundances of ARGs encoding multidrug (oprJ and ttgB genes) and tetracycline (tetV) efflux pump by 608%, 724% and 3052%, respectively. Additionally, we observed an increase in the abundance of numerous bacterial genera under high protistan pressure. Our findings provide empirical evidence that protistan predation significantly promotes antibiotic resistance in soil bacterial communities and advances our understanding of the biological driving forces behind the evolution and development of environmental antibiotic resistance.

Expansion of the cytochrome C oxidase subunit I database and description of four new lobose testate amoebae species (Amoebozoa; Arcellinida).

Arcellinida is ascending in importance in protistology, but description of their diversity still presents multiple challenges. Furthermore, applicable tools for surveillance of these organisms are still in developing stages. Importantly, a good database that sets a correspondence between molecular barcodes and species morphology is lacking. Cytochrome oxidase (COI) has been suggested as the most relevant marker for species discrimination in Arcellinida. However, some major groups of Arcellinida are still lacking a COI sequence. Here we expand the database of COI marker sequences for Arcellinids, using single-cell PCR, transcriptomics, and database scavenging. In the present work, we added 24 new Arcellinida COI sequences to the database, covering all unsampled infra- and suborders. Additionally, we added six new SSUrRNA sequences and described four new species using morphological, morphometrical, and molecular evidence: Heleopera steppica, Centropyxis blatta, Arcella uspiensis, and Cylindrifflugia periurbana. This new database will provide a new starting point to address new research questions from shell evolution, biogeography, and systematics of arcellinids.

Microeukaryotic predators shape the wastewater microbiome.

The physicochemical parameters that shape the prokaryotic community composition in wastewater have been extensively studied. In contrast, it is poorly understood whether and how biotic interactions affect the prokaryotic community composition in wastewater. We used metatranscriptomics data from a bioreactor sampled weekly over 14 months to investigate the wastewater microbiome, including often neglected microeukaryotes. Our analysis revealed that while prokaryotes are unaffected by seasonal changes in water temperature, they are impacted by a seasonal, temperature-induced change in the microeukaryotic community. Our findings suggest that selective predation pressure exerted by microeukaryotes is a significant factor shaping the prokaryotic community in wastewater. This study underscores the importance of investigating the entire wastewater microbiome to develop a comprehensive understanding of wastewater treatment.

Resource-dependent biodiversity and potential multi-trophic interactions determine belowground functional trait stability.

BACKGROUND: For achieving long-term sustainability of intensive agricultural practices, it is pivotal to understand belowground functional stability as belowground organisms play essential roles in soil biogeochemical cycling. It is commonly believed that resource availability is critical for controlling the soil biodiversity and belowground organism interactions that ultimately lead to the stabilization or collapse of terrestrial ecosystem functions, but evidence to support this belief is still limited. Here, we leveraged field experiments from the Chinese National Ecosystem Research Network (CERN) and two microcosm experiments mimicking high and low resource conditions to explore how resource availability mediates soil biodiversity and potential multi-trophic interactions to control functional trait stability. RESULTS: We found that agricultural practice-induced higher resource availability increased potential cross-trophic interactions over 316% in fields, which in turn had a greater effect on functional trait stability, while low resource availability made the stability more dependent on the potential within trophic interactions and soil biodiversity. This large-scale pattern was confirmed by fine-scale microcosm systems, showing that microcosms with sufficient nutrient supply increase the proportion of potential cross-trophic interactions, which were positively associated with functional stability. Resource-driven belowground biodiversity and multi-trophic interactions ultimately feedback to the stability of plant biomass. CONCLUSIONS: Our results indicated the importance of potential multi-trophic interactions in supporting belowground functional trait stability, especially when nutrients are sufficient, and also suggested the ecological benefits of fertilization programs in modern agricultural intensification. Video Abstract.

Plant associated protists-Untapped promising candidates for agrifood tools.

The importance of host-associated microorganisms and their biotic interactions for plant health and performance has been increasingly acknowledged. Protists, main predators and regulators of bacteria and fungi, are abundant and ubiquitous eukaryotes in terrestrial ecosystems. Protists are considered to benefit plant health and performance, but the community structure and functions of plant-associated protists remain surprisingly underexplored. Harnessing plant-associated protists and other microbes can potentially enhance plant health and productivity and sustain healthy food and agriculture systems. In this review, we summarize the knowledge of multifunctionality of protists and their interactions with other microbes in plant hosts, and propose a future framework to study plant-associated protists and utilize protists as agrifood tools for benefiting agricultural production.

What Drives the Assembly of Plant-associated Protist Microbiomes? Investigating the Effects of Crop Species, Soil Type and Bacterial Microbiomes.

In a field experiment we investigated the influence of the environmental filters soil type (i.e. three contrasting soils) and plant species (i.e. lettuce and potato) identity on rhizosphere community assembly of Cercozoa, a dominant group of mostly bacterivorous soil protists. Plant species (14%) and rhizosphere origin (vs bulk soil) with 13%, together explained four times more variation in cercozoan beta diversity than the three soil types (7% explained variation). Our results clearly confirm the existence of plant species-specific protist communities. Network analyses of bacteria-Cercozoa rhizosphere communities identified scale-free small world topologies, indicating mechanisms of self-organization. While the assembly of rhizosphere bacterial communities is bottom-up controlled through the resource supply from root (secondary) metabolites, our results support the hypothesis that the net effect may depend on the strength of top-down control by protist grazers. Since grazing of protists has a strong impact on the composition and functioning of bacteria communities, protists expand the repertoire of plant genes by functional traits, and should be considered as 'protist microbiomes' in analogy to 'bacterial microbiomes'.

Hierarchical phylogenetic community assembly of soil protists in a temperate agricultural field.

Protists are abundant, diverse and perform essential functions in soils. Protistan community structure and its change across time or space are traditionally studied at the species level but the relative importance of the processes shaping these patterns depends on the taxon phylogenetic resolution. Using 18S rDNA amplicon data of the Cercozoa, a group of dominant soil protists, from an agricultural field in western Germany, we observed a turnover of relatively closely related taxa (from sequence variants to genus-level clades) across soil depth; while across soil habitats (rhizosphere, bulk soil, drilosphere), we observed turnover of relatively distantly related taxa, confirming Paracercomonadidae as a rhizosphere-associated clade. We extended our approach to show that closely related Cercozoa encounter divergent arbuscular mycorrhizal (AM) fungi across soil depth and that distantly related Cercozoa encounter closely related AM fungi across soil compartments. This study suggests that soil Cercozoa community assembly at the field scale is driven by niche-based processes shaped by evolutionary legacy of adaptation to conditions primarily related to the soil compartment, followed by the soil layer, giving a deeper understanding on the selection pressures that shaped their evolution.

Phylogenetic analysis confirms the taxonomic placement of the marine flagellate Hermesinum adriaticum (Thecofilosea, Cercozoa, Rhizaria).

Hermesinum adriaticum is a rare marine and brackish flagellate that is of considerable interest due to its markable and fossilizable siliceous skeleton. Based on this skeleton, Hermesinum was initially considered a microalga of the Dictyochophyceae (Ochrophyta, Stramenopiles). Later on, it was assigned to the Ebriida due to its similarity to Ebria tripartita. The taxonomic assignment of the Ebriida, however, changed several times until it was placed within the Thecofilosea (Cercozoa, Rhizaria), based on genetic data of E. tripartita. We sequenced the 18S marker gene sequence of Hermesinum and confirm the close relationship of Ebria and Hermesinum.

Microeukaryotic gut parasites in wastewater treatment plants: diversity, activity, and removal.

BACKGROUND: During wastewater treatment, the wastewater microbiome facilitates the degradation of organic matter, reduction of nutrients, and removal of gut parasites. While the latter function is essential to minimize public health risks, the range of parasites involved and how they are removed is still poorly understood. RESULTS: Using shotgun metagenomic (DNA) and metatranscriptomic (RNA) sequencing data from ten wastewater treatment plants in Switzerland, we were able to assess the entire wastewater microbiome, including the often neglected microeukaryotes (protists). In the latter group, we found a surprising richness and relative abundance of active parasites, particularly in the inflow. Using network analysis, we tracked these taxa across the various treatment compartments and linked their removal to trophic interactions. CONCLUSIONS: Our results indicate that the combination of DNA and RNA data is essential for assessing the full spectrum of taxa present in wastewater. In particular, we shed light on an important but poorly understood function of wastewater treatment - parasite removal. Video Abstract.

Transfer of the thecate amoebae Lecythium spinosum and Pamphagus armatus to Rhizaspis (Thecofilosea, Cercozoa, Rhizaria).

Thecofilosea is a class in Cercozoa (Rhizaria) comprising mainly freshwater-inhabiting algivores. Recently, numerous isolates of thecofilosean amoebae have been cultured and were characterized by an integrated morphological and molecular approach. The captivating spine-bearing taxa in Thecofilosea were not yet molecularly characterized due to being very rare. There are only two known spine-bearing species, Pamphagus armatus and Lecythium spinosum, which were synonymized by Penard in 1902. Due to a morphological difference of those taxa, we discuss here that we disagree with this taxonomical act. We further isolated single cells of Pamphagus armatus directly from their habitat and successfully sequenced their SSU rDNA, which we subjected to phylogenetic analyses. We show that Pamphagus armatus branches within the Rhizaspididae (Tectofilosida, Thecofilosea). Accordingly, we transfer Pamphagus armatus and the assumingly closely related species Lecythium spinosum to Rhizaspis.

Eukaryotic rather than prokaryotic microbiomes change over seasons in rewetted fen peatlands.

In the last decades, rewetting of drained peatlands is on the rise worldwide, to restore their significant carbon sink function. Despite the increasing understanding of peat microbiomes, little is known about the seasonal dynamics and network interactions of the microbial communities in these ecosystems, especially in rewetted fens (groundwater-fed peatlands). Here, we investigated the seasonal dynamics in both prokaryotic and eukaryotic microbiomes in three common fen types in Northern Germany. The eukaryotic microbiomes, including fungi, protists and microbial metazoa, showed significant changes in their community structures across the seasons in contrast to largely unaffected prokaryotic microbiomes. Furthermore, our results proved that the dynamics in eukaryotic microbiomes in the rewetted sites differed between fen types, specifically in terms of saprotrophs, arbuscular mycorrhiza and grazers of bacteria. The co-occurrence networks also exhibited strong seasonal dynamics that differed between rewetted and drained sites, and the correlations involving protists and prokaryotes were the major contributors to these dynamics. Our study provides the insight that microbial eukaryotes mainly define the seasonal dynamics of microbiomes in rewetted fen peatlands. Accordingly, future research should unravel the importance of eukaryotes for biogeochemical processes, especially the under-characterized protists and metazoa, in these poorly understood ecosystems.

The wastewater protist Rhogostoma minus (Thecofilosea, Rhizaria) is abundant, widespread, and hosts Legionellales.

Wastewater is treated by concerted actions of the microbial communities within bioreactors. Although protists (unicellular eukaryotes) are good bioindicators and important players influencing denitrification, nitrification, and flocculation, they are the least known organisms in WWTPs. The few recent environmental surveys of the protistan diversity in WWTPs show that the most abundant protistan sequences in WWTPs belong to Thecofilosea (Rhizaria). We re-investigated previously published environmental sequencing data and gathered strains from seven WWTPs to determine which species dominate WWTPs worldwide. We found that all highly abundant thecofilosean sequences represent a single species - Rhogostoma minus. Considering that Thecofilosea are frequent hosts for Legionellales, i.e. bacteria linked to waterborne diseases, we confirm that Rhogostoma minus functions as a host for Legionellales in WWTPs. Whether the highly abundant Rhogostoma minus also serves as a host for known human pathogenic Legionellales requires further attention.

On the phenology of protists: recurrent patterns reveal seasonal variation of protistan (Rhizaria: Cercozoa and Endomyxa) communities in tree canopies.

Tree canopies are colonized by billions of highly specialized microorganisms that are well adapted to the highly variable microclimatic conditions, caused by diurnal fluctuations and seasonal changes. In this study, we investigated seasonality patterns of protists in the tree canopies of a temperate floodplain forest via high-throughput sequencing with group-specific primers for the phyla Cercozoa and Endomyxa. We observed consistent seasonality, and identified divergent spring and autumn taxa. Tree crowns were characterized by a dominance of bacterivores and omnivores, while eukaryvores gained a distinctly larger share in litter and soil communities on the ground. In the canopy seasonality was largest among communities detected on the foliar surface: In spring, higher variance within alpha diversity of foliar samples indicated greater heterogeneity during initial colonization. However, communities underwent compositional changes during the aging of leaves in autumn, highly reflecting recurring phenological changes during protistan colonization. Surprisingly, endomyxan root pathogens appeared to be exceptionally abundant across tree canopies during autumn, demonstrating a potential role of the canopy surface as a physical filter for air-dispersed propagules. Overall, about 80% of detected OTUs could not be assigned to known species-representing dozens of microeukaryotic taxa whose canopy inhabitants are waiting to be discovered.

Assembly Patterns of the Rhizosphere Microbiome Along the Longitudinal Root Axis of Maize (Zea mays L.).

It is by now well proven that different plant species within their specific root systems select for distinct subsets of microbiota from bulk soil - their individual rhizosphere microbiomes. In maize, root growth advances several centimeters each day, with the locations, quality and quantity of rhizodeposition changing. We investigated the assembly of communities of prokaryotes (archaea and bacteria) and their protistan predators (Cercozoa, Rhizaria) along the longitudinal root axis of maize (Zea mays L.). We grew maize plants in an agricultural loamy soil and sampled rhizosphere soil at distinct locations along maize roots. We applied high-throughput sequencing, followed by diversity and network analyses in order to track changes in relative abundances, diversity and co-occurrence of rhizosphere microbiota along the root axis. Apart from a reduction of operational taxonomic unit (OTU) richness and a strong shift in community composition between bulk soil and root tips, patterns of microbial community assembly along maize-roots were more complex than expected. High variation in beta diversity at root tips and the root hair zone indicated substantial randomness of community assembly. Root hair zone communities were characterized by massive co-occurrence of microbial taxa, likely fueled by abundant resource supply from rhizodeposition. Further up the root where lateral roots emerged processes of community assembly appeared to be more deterministic (e.g., through competition and predation). This shift toward significance of deterministic processes was revealed by low variability of beta diversity, changes in network topology, and the appearance of regular phylogenetic co-occurrence patterns in bipartite networks between prokaryotes and their potential protistan predators. Such patterns were strongest in regions with fully developed laterals, suggesting that a consistent rhizosphere microbiome finally assembled. For the targeted improvement of microbiome function, such knowledge on the processes of microbiome assembly on roots and its temporal and spatial variability is crucially important.

Novel Endosymbionts in Rhizarian Amoebae Imply Universal Infection of Unrelated Free-Living Amoebae by Legionellales.

Legionellales-infected water is a frequent cause of local outbreaks of Legionnaires' disease and Pontiac fever. Decontaminations are difficult because Legionellales reproduce in eukaryotic microorganisms (protists). Most often, Legionellales have been isolated from amoebae; however, the culture-based sampling methods are taxonomically biased. Sequencing studies show that amoebae in the cercozoan class Thecofilosea are dominant in soils and wastewater treatment plants, prompting us to screen their capability to serve as potential hosts of endosymbiotic bacteria. Environmental isolates of Thecofilosea contained a surprising richness of endosymbiotic Legionellales, including Legionella. Considering the widespread dispersal of Legionellales in apparently unrelated amoeboid protist taxa, it appears that the morphotype and not the evolutionary origin of amoebae determines their suitability as hosts for Legionellales. We further provide a protocol for gnotobiotic cultivation of Legionellales and their respective hosts, facilitating future genomic and transcriptomic research of host-symbiont relationships.

Taxonomic and Functional Diversity of Heterotrophic Protists (Cercozoa and Endomyxa) from Biological Soil Crusts.

Biological soil crusts (biocrusts) accommodate diverse communities of phototrophic and heterotrophic microorganisms. Heterotrophic protists have critical roles in the microbial food webs of soils, with Cercozoa and Endomyxa often being dominant groups. Still, the diversity, community composition, and functions of Cercozoa and Endomyxa in biocrusts have been little explored. In this study, using a high-throughput sequencing method with taxon-specific barcoded primers, we studied cercozoan and endomyxan communities in biocrusts from two unique habitats (subarctic grassland and temperate dunes). The communities differed strongly, with the grassland and dunes being dominated by Sarcomonadea (69%) and Thecofilosea (43%), respectively. Endomyxa and Phytomyxea were the minor components in dunes. Sandonidae, Allapsidae, and Rhogostomidae were the most abundant taxa in both habitats. In terms of functionality, up to 69% of the grassland community was constituted by bacterivorous Cercozoa. In contrast, cercozoan and endomyxan communities in dunes consisted of 31% bacterivores, 25% omnivores, and 20% eukaryvores. Facultative and obligate eukaryvores mostly belonged to the families Rhogostomidae, Fiscullidae, Euglyphidae, Leptophryidae, and Cercomonadidae, most of which are known to feed mainly on algae. Biocrust edaphic parameters such as pH, total organic carbon, nitrogen, and phosphorus did not have any significant influence on shaping cercozoan communities within each habitat, which confirms previous results from dunes.