Divergent impacts of fertilization regimes on below-ground prokaryotic and eukaryotic communities in the Tibetan Plateau.

Chemical nutrient amendment by human activities can lead to environmental impacts contributing to global biodiversity loss. However, the comprehensive understanding of how below- and above-ground biodiversity shifts under fertilization regimes in natural ecosystems remains elusive. Here, we conducted a seven-year field experiment (2011-2017) and examined the effects of different fertilization on plant biodiversity and soil belowground (prokaryotic and eukaryotic) communities in the alpine meadow of the Tibetan Plateau, based on data collected in 2017. Our results indicate that nitrogen addition promoted total plant biomass but reduced the plant species richness. Conversely, phosphorus enrichment did not promote plant biomass and exhibited an unimodal pattern with plant richness. In the belowground realm, distinct responses of soil prokaryotic and eukaryotic communities were observed under fertilizer application. Specifically, soil prokaryotic diversity decreased with nitrogen enrichment, correlating with shifts in soil pH. Similarly, soil eukaryotic diversity decreased with increased phosphorous inputs, aligning with the equilibrium between soil available and total phosphorus. We also established connections between these soil organism communities with above-ground plant richness and biomass. Overall, our study contributes to a better understanding of the sustainable impacts of human-induced nutrient enrichment on the natural environment. Future research should delve deeper into the long-term effects of fertilization on soil health and ecosystem functioning, aiming to achieve a balance between agricultural productivity and environmental conservation.

Rhizosphere shapes the associations between protistan predators and bacteria within microbiomes through the deterministic selection on bacterial communities.

The assembly of bacterial communities in the rhizosphere is well-documented and plays a crucial role in supporting plant performance. However, we have limited knowledge of how plant rhizosphere determines the assembly of protistan predators and whether the potential associations between protistan predators and bacterial communities shift due to rhizosphere selection. To address this, we examined bacterial and protistan taxa from 443 agricultural soil samples including bulk and rhizosphere soils. Our results presented distinct patterns of bacteria and protistan predators in rhizosphere microbiome assembly. Community assembly of protistan predators was determined by a stochastic process in the rhizosphere and the diversity of protistan predators was reduced in the rhizosphere compared to bulk soils, these may be attributed to the indirect impacts from the altered bacterial communities that showed deterministic process assembly in the rhizosphere. Interestingly, we observed that the plant rhizosphere facilitates more close interrelationships between protistan predators and bacterial communities, which might promote a healthy rhizosphere microbial community for plant growth. Overall, our findings indicate that the potential predator-prey relationships within the microbiome, mediated by plant rhizosphere, might contribute to plant performance in agricultural ecosystems.

Foliar Spraying of Chlorpyrifos Triggers Plant Production of Linolenic Acid Recruiting Rhizosphere Bacterial Sphingomonas sp.

Plants have developed an adaptive strategy for coping with biotic or abiotic stress by recruiting specific microorganisms from the soil pool. Recent studies have shown that the foliar spraying of pesticides causes oxidative stress in plants and leads to changes in the rhizosphere microbiota, but the mechanisms by which these microbiota change and rebuild remain unclear. Herein, we provide for the first-time concrete evidence that rice plants respond to the stress of application of the insecticide chlorpyrifos (CP) by enhancing the release of amino acids, lipids, and nucleotides in root exudates, leading to a shift in rhizosphere bacterial community composition and a strong enrichment of the genus Sphingomonas sp. In order to investigate the underlying mechanisms, we isolated a Sphingomonas representative isolate and demonstrated that it is both attracted by and able to consume linolenic acid, one of the root exudates overproduced after pesticide application. We further show that this strain selectively colonizes roots of treated plants and alleviates pesticide stress by degrading CP and releasing plant-beneficial metabolites. These results indicate a feedback loop between plants and their associated microbiota allowing to respond to pesticide-induced stress.

Effect of mutation supply on population dynamics and trait evolution in an experimental microbial community.

Mutation supply can influence evolutionary and thereby ecological dynamics in important ways which have received little attention. Mutation supply influences features of population genetics, such as the pool of adaptive mutations, evolutionary pathways and importance of processes, such as clonal interference. The resultant trait evolutionary dynamics, in turn, can alter population size and species interactions. However, controlled experiments testing for the importance of mutation supply on rapid adaptation and thereby population and community dynamics have primarily been restricted to the first of these aspects. To close this knowledge gap, we performed a serial passage experiment with wild-type Pseudomonas fluorescens and a mutant with reduced mutation rate. Bacteria were grown at two resource levels in combination with the presence of a ciliate predator. A higher mutation supply enabled faster adaptation to the low-resource environment and anti-predatory defence. This was associated with higher population size at the ecological level and better access to high-recurrence mutational targets at the genomic level with higher mutation supply. In contrast, mutation rate did not affect growth under high-resource level. Our results demonstrate that intrinsic mutation rate influences population dynamics and trait evolution particularly when population size is constrained by extrinsic conditions.

Resource availability drives bacteria community resistance to pathogen invasion via altering bacterial pairwise interactions.

Microbial interactions within resident communities are a major determinant of resistance to pathogen invasion. Yet, interactions vary with environmental conditions, raising the question of how community composition and environments interactively shape invasion resistance. Here, we use resource availability (RA) as a model parameter altering the resistance of model bacterial communities to invasion by the plant pathogenic bacterium Ralstonia solanacearum. We found that at high RA, interactions between resident bacterial species were mainly driven by the direct antagonism, in terms of the means of invader inhibition. Consequently, the competitive resident communities with a higher production of antibacterial were invaded to a lesser degree than facilitative communities. At low RA, bacteria produced little direct antagonist potential, but facilitative communities reached a relatively higher community productivity, which showed higher resistance to pathogen invasion than competitive communities with lower productivities. This framework may lay the basis to understand complex microbial interactions and biological invasion as modulated by the dynamic changes of environmental resource availability.

Introduction of probiotic bacterial consortia promotes plant growth via impacts on the resident rhizosphere microbiome.

Plant growth depends on a range of functions provided by their associated rhizosphere microbiome, including nutrient mineralization, hormone co-regulation and pathogen suppression. Improving the ability of plant-associated microbiomes to deliver these functions is thus important for developing robust and sustainable crop production. However, it is yet unclear how beneficial effects of probiotic microbial inoculants can be optimized and how their effects are mediated. Here, we sought to enhance tomato plant growth by targeted introduction of probiotic bacterial consortia consisting of up to eight plant-associated Pseudomonas strains. We found that the effect of probiotic consortium inoculation was richness-dependent: consortia that contained more Pseudomonas strains reached higher densities in the tomato rhizosphere and had clearer beneficial effects on multiple plant growth characteristics. Crucially, these effects were best explained by changes in the resident community diversity, composition and increase in the relative abundance of initially rare taxa, instead of introduction of plant-beneficial traits into the existing community along with probiotic consortia. Together, our results suggest that beneficial effects of microbial introductions can be driven indirectly through effects on the diversity and composition of the resident plant rhizosphere microbiome.

Targeted plant hologenome editing for plant trait enhancement.

Breeding better crops is a cornerstone of global food security. While efforts in plant genetic improvement show promise, it is increasingly becoming apparent that the plant phenotype should be treated as a function of the holobiont, in which plant and microbial traits are deeply intertwined. Using a minimal holobiont model, we track ethylene production and plant nutritional value in response to alterations in plant ethylene synthesis (KO mutation in ETO1), which induces 1-aminocyclopropane-1-carboxylic acid (ACC) synthase 5 (ACS5), or microbial degradation of ACC (KO mutation in microbial acdS), preventing the breakdown of the plant ACC pool, the product of ACS5. We demonstrate that similar plant phenotypes can be generated by either specific mutations of plant-associated microbes or alterations in the plant genome. Specifically, we could equally increase plant nutritional value by either altering the plant ethylene synthesis gene ETO1, or the microbial gene acdS. Both mutations yielded a similar plant phenotype with increased ethylene production and higher shoot micronutrient concentrations. Restoring bacterial AcdS enzyme activity also rescued the plant wild-t8yp phenotype in an eto1 background. Plant and bacterial genes build an integrated plant-microbe regulatory network amenable to genetic improvement from both the plant and microbial sides.

Rhizosphere microbiome functional diversity and pathogen invasion resistance build up during plant development.

The rhizosphere microbiome is essential for plant growth and health, and numerous studies have attempted to link microbiome functionality to species and trait composition. However, to date little is known about the actual ecological processes shaping community composition, complicating attempts to steer microbiome functionality. Here, we assess the development of microbial life history and community-level species interaction patterns that emerge during plant development. We use microbial phenotyping to experimentally test the development of niche complementarity and life history traits linked to microbiome performance. We show that the rhizosphere microbiome assembles from pioneer assemblages of species with random resource overlap into high-density, functionally complementary climax communities at later stages. During plant growth, fast-growing species were further replaced by antagonistic and stress-tolerant ones. Using synthetic consortia isolated from different plant growth stages, we demonstrate that the high functional diversity of 'climax' microbiomes leads to a better resistance to bacterial pathogen invasion. By demonstrating that different life-history strategies prevail at different plant growth stages and that community-level processes may supersede the importance of single species, we provide a new toolbox to understand microbiome assembly and steer its functionality at a community level.

Bacterial community richness shifts the balance between volatile organic compound-mediated microbe-pathogen and microbe-plant interactions.

Even though bacteria are important in determining plant growth and health via volatile organic compounds (VOCs), it is unclear how these beneficial effects emerge in multi-species microbiomes. Here we studied this using a model plant-bacteria system, where we manipulated bacterial community richness and composition and determined the subsequent effects on VOC production and VOC-mediated pathogen suppression and plant growth-promotion. We assembled VOC-producing bacterial communities in different richness levels ranging from one to 12 strains using three soil-dwelling bacterial genera (Bacillus, Paenibacillus and Pseudomonas) and investigated how the composition and richness of bacterial community affect the production and functioning of VOCs. We found that VOC production correlated positively with pathogen suppression and plant growth promotion and that all bacteria produced a diverse set of VOCs. However, while pathogen suppression was maximized at intermediate community richness levels when the relative amount and the number of VOCs were the highest, plant growth promotion was maximized at low richness levels and was only affected by the relative amount of plant growth-promoting VOCs. The contrasting effects of richness could be explained by differences in the amount and number of produced VOCs and by opposing effects of community productivity and evenness on pathogen suppression and plant-growth promotion along the richness gradient. Together, these results suggest that the number of interacting bacterial species and the structure of the rhizosphere microbiome drive the balance between VOC-mediated microbe-pathogen and microbe-plant interactions potentially affecting plant disease outcomes in natural and agricultural ecosystems.

Facilitation promotes invasions in plant-associated microbial communities.

While several studies have established a positive correlation between community diversity and invasion resistance, it is less clear how species interactions within resident communities shape this process. Here, we experimentally tested how antagonistic and facilitative pairwise interactions within resident model microbial communities predict invasion by the plant-pathogenic bacterium Ralstonia solanacearum. We found that facilitative resident community interactions promoted and antagonistic interactions suppressed invasions both in the lab and in the tomato plant rhizosphere. Crucially, pairwise interactions reliably explained observed invasion outcomes also in multispecies communities, and mechanistically, this was linked to direct inhibition of the invader by antagonistic communities (antibiosis), and to a lesser degree by resource competition between members of the resident community and the invader. Together, our findings suggest that the type and strength of pairwise interactions can reliably predict the outcome of invasions in more complex multispecies communities.

Resource stoichiometry shapes community invasion resistance via productivity-mediated species identity effects.

Diversity-invasion resistance relationships are often variable and sensitive to environmental conditions such as resource availability. Resource stoichiometry, the relative concentration of different elements in the environment, has been shown to have strong effects on the physiology and interactions between different species. Yet, its role for diversity-invasion resistance relationships is still poorly understood. Here, we explored how the ratio of nitrogen (N) and phosphorus affects the productivity and invasion resistance of constructed microbial communities by a plant pathogenic bacterium, Ralstonia solanacearum. We found that resource stoichiometry and species identity effects affected the invasion resistance of communities. Both high N concentration and resident community diversity constrained invasions, and two resident species, in particular, had strong negative effects on the relative density of the invader and the resident community productivity. While resource stoichiometry did not affect the mean productivity of the resident community, it favoured the growth of two species that strongly constrained invasions turning the slope of productivity-invasion resistance relationship more negative. Together our findings suggest that alterations in resource stoichiometry can change the community resistance to invasions by having disproportionate effects on species growth, potentially explaining changes in microbial community composition under eutrophication.

Resource availability modulates biodiversity-invasion relationships by altering competitive interactions.

Community diversity affects the survival of newly introduced species via resource competition. Competitive interactions can be modulated by resource availability and we hypothesized that this may alter biodiversity-invasion relationships. To study this, we assessed the growth of a bacterial invader, Ralstonia solanacearum, when introduced into communities comprised of one to five closely related resident species under different resource concentrations. The invader growth was then examined as a function of resident community richness, species composition and resource availability. We found that the relative density of the invader was reduced by increasing resident community richness and resource availability. Mechanistically, this could be explained by changes in the competitive interactions between the resident species and the invader along the resource availability gradient. At low resource availability, resident species with a high catabolic similarity with the invader efficiently reduced the invader relative density, while at high resource availability, fast-growing resident species became more important for the invader suppression. These results indicate that the relative importance of different resident community species can change dynamically along to resource availability gradient. Diverse communities could be thus more robust to invasions by providing a set of significant species that can take suppressive roles across different environments.

Coprophilic amoebae and flagellates, including Guttulinopsis, Rosculus and Helkesimastix, characterise a divergent and diverse rhizarian radiation and contribute to a large diversity of faecal-associated protists.

A wide diversity of organisms utilize faecal habitats as a rich nutrient source or a mechanism to traverse through animal hosts. We sequenced the 18S rRNA genes of the coprophilic, fruiting body-forming amoeba Guttulinopsis vulgaris and its non-fruiting relatives Rosculus 'ithacus' CCAP 1571/3, R. terrestris n. sp. and R. elongata n. sp. and demonstrate that they are related to the coprophilic flagellate Helkesimastix in a strongly supported, but highly divergent 18S sister clade. PCR primers specific to both clades were used to generate 18S amplicons from a range of environmental and faecal DNA samples. Phylogenetic analysis of the cloned sequences demonstrated a high diversity of uncharacterised sequence types within this clade, likely representing previously described members of the genera Guttulinopsis, Rosculus and Helkesimastix, as well as so-far unobserved organisms. Further, an Illumina MiSeq sequenced set of 18S V4-region amplicons generated from faecal DNAs using universal eukaryote primers showed that core-cercozoan assemblages in faecal samples are as diverse as those found in more conventionally examined habitats. These results reveal many novel lineages, some of which appear to occur preferentially in faecal material, in particular cercomonads and glissomonads. More broadly, we show that faecal habitats are likely untapped reservoirs of microbial eukaryotic diversity.

Biodiversity and species identity shape the antifungal activity of bacterial communities.

Soils host diverse communities of interacting microbes and the nature of interspecific interactions is increasingly recognized to affect ecosystem-level processes. Antagonistic interactions between bacteria and fungi are of particular relevance for soil functioning. A number of soil bacteria produce secondary metabolites that inhibit eukaryotic growth. Antibiosis may be stimulated in the presence of competing bacteria, and we tested if biodiversity within bacterial communities affects their antagonistic activity against fungi and fungal-like species. We set up Pseudomonas communities of increasing diversity and measured the production of the broad spectrum antifungal compound 2,4-DAPG and their antagonistic activity against different eukaryotes. Diversity increased DAPG concentration and antifungal activity, an effect due to a combination of identity and interactions between species. Our results indicate that investment of pseudomonads into broad spectrum anti-eukaryotic traits is determined by both community composition and diversity and this provides new avenues to understand interactions between bacterial and fungal communities.

Microbe-induced phenotypic variation leads to overyielding in clonal plant populations.

Overyielding, the high productivity of multispecies plant communities, is commonly seen as the result of plant genetic diversity. Here we demonstrate that biodiversity-ecosystem functioning relationships can emerge in clonal plant populations through interaction with microorganisms. Using a model clonal plant species, we found that exposure to volatiles of certain microorganisms led to divergent plant phenotypes. Assembling communities out of plants associated with different microorganisms led to transgressive overyielding in both biomass and seed yield. Our results highlight the importance of belowground microbial diversity in plant biodiversity research and open new avenues for precision ecosystem management.

Synthetic microbial communities: Sandbox and blueprint for soil health enhancement.

We summarize here the use of SynComs in improving various dimensions of soil health, including fertility, pollutant removal, soil-borne disease suppression, and soil resilience; as well as a set of useful guidelines to assess and understand the principles for designing SynComs to enhance soil health. Finally, we discuss the next stages of SynComs applications, including highly diverse and multikingdom SynComs targeting several functions simultaneously.

Pollutant profile complexity governs wastewater removal of recalcitrant pharmaceuticals.

Organic pollutants are an increasing threat for wildlife and humans. Managing their removal is however complicated by the difficulties in predicting degradation rates. In this work, we demonstrate that the complexity of the pollutant profile, the set of co-existing contaminants, is a major driver of biodegradation in wastewater. We built representative assemblages out of one to five common pharmaceuticals (caffeine, atenolol, paracetamol, ibuprofen, and enalapril) selected along a gradient of biodegradability. We followed their individual removal by wastewater microbial communities. The presence of multichemical background pollution was essential for the removal of recalcitrant molecules such as ibuprofen. High-order interactions between multiple pollutants drove removal efficiency. We explain these interactions by shifts in the microbiome, with degradable molecules such as paracetamol enriching species and pathways involved in the removal of several organic pollutants. We conclude that pollutants should be treated as part of a complex system, with emerging pollutants potentially showing cascading effects and offering leverage to promote bioremediation.

Effects of plant tissue permeability on invasion and population bottlenecks of a phytopathogen.

Pathogen genetic diversity varies in response to environmental changes. However, it remains unclear whether plant barriers to invasion could be considered a genetic bottleneck for phytopathogen populations. Here, we implement a barcoding approach to generate a pool of 90 isogenic and individually barcoded Ralstonia solanacearum strains. We used 90 of these strains to inoculate tomato plants with different degrees of physical permeability to invasion (intact roots, wounded roots and xylem inoculation) and quantify the phytopathogen population dynamics during invasion. Our results reveal that the permeability of plant roots impacts the degree of population bottleneck, genetic diversity, and composition of Ralstonia populations. We also find that selection is the main driver structuring pathogen populations when barriers to infection are less permeable, i.e., intact roots, the removal of root physical and immune barriers results in the predominance of stochasticity in population assembly. Taken together, our study suggests that plant root permeability constitutes a bottleneck for phytopathogen invasion and genetic diversity.

Stable isotope probing and Raman spectroscopy for monitoring carbon flow in a food chain and revealing metabolic pathway.

Accurately measuring carbon flows is a challenge for understanding processes such as diverse intracellular metabolic pathways and predator-prey interactions. Combined with stable isotope probing (SIP), single-cell Raman spectroscopy was demonstrated for the first time to link the food chain from carbon substrate to bacterial prey up to predators at the single-cell level in a quantitative and nondestructive manner. Escherichia coli OP50 with different (13)C content, which were grown in a mixture of (12)C- and fully carbon-labeled (13)C-glucose (99%) as a sole carbon source, were fed to the nematode. The (13)C signal in Caenorhabditis elegans was proportional to the (13)C content in E. coli. Two Raman spectral biomarkers (Raman bands for phenylalanine at 1001 cm(-1) and thymine at 747 cm(-1) Raman bands), were used to quantify the (13)C content in E. coli and C. elegans over a range of 1.1-99%. The phenylalanine Raman band was a suitable biomarker for prokaryotic cells and thymine Raman band for eukaryotic cells. A biochemical mechanism accounting for the Raman red shifts of phenylalanine and thymine in response to (13)C-labeling is proposed in this study and is supported by quantum chemical calculation. This study offers new insights of carbon flow via the food chain and provides a research tool for microbial ecology and investigation of biochemical pathways.

Bacterial volatile organic compounds attenuate pathogen virulence via evolutionary trade-offs.

Volatile organic compounds (VOCs) produced by soil bacteria have been shown to exert plant pathogen biocontrol potential owing to their strong antimicrobial activity. While the impact of VOCs on soil microbial ecology is well established, their effect on plant pathogen evolution is yet poorly understood. Here we experimentally investigated how plant-pathogenic Ralstonia solanacearum bacterium adapts to VOC-mixture produced by a biocontrol Bacillus amyloliquefaciens T-5 bacterium and how these adaptations might affect its virulence. We found that VOC selection led to a clear increase in VOC-tolerance, which was accompanied with cross-tolerance to several antibiotics commonly produced by soil bacteria. The increasing VOC-tolerance led to trade-offs with R. solanacearum virulence, resulting in almost complete loss of pathogenicity in planta. At the genetic level, these phenotypic changes were associated with parallel mutations in genes encoding lipopolysaccharide O-antigen (wecA) and type-4 pilus biosynthesis (pilM), which both have been linked with outer membrane permeability to antimicrobials and plant pathogen virulence. Reverse genetic engineering revealed that both mutations were important, with pilM having a relatively larger negative effect on the virulence, while wecA having a relatively larger effect on increased antimicrobial tolerance. Together, our results suggest that microbial VOCs are important drivers of bacterial evolution and could potentially be used in biocontrol to select for less virulent pathogens via evolutionary trade-offs.