Size exclusion experiment in a grassland field unravels top-down control of the soil fauna on microbial community assembly.

Highly diverse and abundant organisms coexist in soils. However, the contribution of biotic interactions between soil organisms to microbial community assembly remains to be explored. Here, we assess the extent to which soil fauna can shape microbial community assembly using an exclusion experiment in a grassland field to sort soil biota based on body size. After 1 year, the exclusion of larger fauna favoured phagotrophic protists, with increases up to 32% in their proportion compared to the no-mesh treatment. In contrast, members of the bacterial community and to a lesser extent of the fungal community were negatively impacted. Shifts in bacterial but not in fungal communities were best explained by the response of the protistan community to exclusion. Our findings provide empirical evidence of top-down control on the soil microbial communities and underline the importance of integrating higher trophic levels for a better understanding of the soil microbiome assembly.

Engineering multi-degrading bacterial communities to bioremediate soils contaminated with pesticides residues.

Parallel to the important use of pesticides in conventional agriculture there is a growing interest for green technologies to clear contaminated soil from pesticides and their degradation products. Bioaugmentation i. e. the inoculation of degrading micro-organisms in polluted soil, is a promising method still in needs of further developments. Specifically, improvements in the understanding of how degrading microorganisms must overcome abiotic filters and interact with the autochthonous microbial communities are needed in order to efficiently design bioremediation strategies. Here we designed a protocol aiming at studying the degradation of two herbicides, glyphosate (GLY) and isoproturon (IPU), via experimental modifications of two source bacterial communities. We used statistical methods stemming from genomic prediction to link community composition to herbicides degradation potentials. Our approach proved to be efficient with correlation estimates over 0.8 - between model predictions and measured pesticide degradation values. Multi-degrading bacterial communities were obtained by coalescing bacterial communities with high GLY or IPU degradation ability based on their community-level properties. Finally, we evaluated the efficiency of constructed multi-degrading communities to remove pesticide contamination in a different soil. While results are less clear in the case of GLY, we showed an efficient transfer of degrading capacities towards the receiving soil even at relatively low inoculation levels in the case of IPU. Altogether, we developed an innovative protocol for building multi-degrading simplified bacterial communities with the help of genomic prediction tools and coalescence, and proved their efficiency in a contaminated soil.

Manipulating the physical distance between cells during soil colonization reveals the importance of biotic interactions in microbial community assembly.

BACKGROUND: Microbial communities are of tremendous importance for ecosystem functioning and yet we know little about the ecological processes driving the assembly of these communities in the environment. Here, we used an unprecedented experimental approach based on the manipulation of physical distance between neighboring cells during soil colonization to determine the role of bacterial interactions in soil community assembly. We hypothesized that experimentally manipulating the physical distance between bacterial cells will modify the interaction strengths leading to differences in microbial community composition, with increasing distance between neighbors favoring poor competitors. RESULTS: We found significant differences in both bacterial community diversity, composition and co-occurrence networks after soil colonization that were related to physical distancing. We show that reducing distances between cells resulted in a loss of bacterial diversity, with at least 41% of the dominant OTUs being significantly affected by physical distancing. Our results suggest that physical distancing may differentially modulate competitiveness between neighboring species depending on the taxa present in the community. The mixing of communities that assembled at high and low cell densities did not reveal any "home field advantage" during coalescence. This confirms that the observed differences in competitiveness were due to biotic rather than abiotic filtering. CONCLUSIONS: Our study demonstrates that the competitiveness of bacteria strongly depends on cell density and community membership, therefore highlighting the fundamental role of microbial interactions in the assembly of soil communities.

Deciphering the biotic and abiotic drivers of coalescence asymmetry between soil and manure microbiomes.

Manure application improves soil fertility, yet its implications on the success of invasion of manure-borne microorganisms in the soil are poorly understood. Here, we assessed the importance of abiotic and biotic factors in modulating the extent to which manure-borne fungal and bacterial communities can invade resident soil microbial communities. For this purpose, we applied varying frequencies of two different amounts of manure to nine soils differing in physico-chemical properties, as well as in land-use history, over 180 days and monitored changes in bacterial and fungal communities. Variance partitioning revealed differential contributions of abiotic and biotic factors to invasion success, which together accounted for up to 82 % of the variance explained. We showed that the effects of interactions between biotic and abiotic factors increased with coalescence frequency and manure amount for the bacterial and fungal communities, respectively. Both abiotic and biotic factors were important for modulating coalescence asymmetry for the bacterial community, whereas abiotic factors had a greater effect on the fungal community. These results provide new insights into the drivers of coalescence events between manure and resident soil microbial communities. Moreover, our findings highlight the roles of the mixing ratio and frequency of coalescence events in modulating the survival of manure-borne microorganisms.

Biotic homogenization, lower soil fungal diversity and fewer rare taxa in arable soils across Europe.

Soil fungi are a key constituent of global biodiversity and play a pivotal role in agroecosystems. How arable farming affects soil fungal biogeography and whether it has a disproportional impact on rare taxa is poorly understood. Here, we used the high-resolution PacBio Sequel targeting the entire ITS region to investigate the distribution of soil fungi in 217 sites across a 3000 km gradient in Europe. We found a consistently lower diversity of fungi in arable lands than grasslands, with geographic locations significantly impacting fungal community structures. Prevalent fungal groups became even more abundant, whereas rare groups became fewer or absent in arable lands, suggesting a biotic homogenization due to arable farming. The rare fungal groups were narrowly distributed and more common in grasslands. Our findings suggest that rare soil fungi are disproportionally affected by arable farming, and sustainable farming practices should protect rare taxa and the ecosystem services they support.

Experimental community coalescence sheds light on microbial interactions in soil and restores impaired functions.

BACKGROUND: Microbes typically live in communities where individuals can interact with each other in numerous ways. However, knowledge on the importance of these interactions is limited and derives mainly from studies using a limited number of species grown in coculture. Here, we manipulated soil microbial communities to assess the contribution of interactions between microorganisms for assembly of the soil microbiome. RESULTS: By combining experimental removal (taxa depletion in the community) and coalescence (mixing of manipulated and control communities) approaches, we demonstrated that interactions between microorganisms can play a key role in determining their fitness during soil recolonization. The coalescence approach not only revealed the importance of density-dependent interactions in microbial community assembly but also allowed to restore partly or fully community diversity and soil functions. Microbial community manipulation resulted in shifts in both inorganic nitrogen pools and soil pH, which were related to the proportion of ammonia-oxidizing bacteria. CONCLUSIONS: Our work provides new insights into the understanding of the importance of microbial interactions in soil. Our top-down approach combining removal and coalescence manipulation also allowed linking community structure and ecosystem functions. Furthermore, these results highlight the potential of manipulating microbial communities for the restoration of soil ecosystems. Video Abstract.

The impact of agricultural management on soil aggregation and carbon storage is regulated by climatic thresholds across a 3000 km European gradient.

Organic carbon and aggregate stability are key features of soil quality and are important to consider when evaluating the potential of agricultural soils as carbon sinks. However, we lack a comprehensive understanding of how soil organic carbon (SOC) and aggregate stability respond to agricultural management across wide environmental gradients. Here, we assessed the impact of climatic factors, soil properties and agricultural management (including land use, crop cover, crop diversity, organic fertilization, and management intensity) on SOC and the mean weight diameter of soil aggregates, commonly used as an indicator for soil aggregate stability, across a 3000 km European gradient. Soil aggregate stability (-56%) and SOC stocks (-35%) in the topsoil (20 cm) were lower in croplands compared with neighboring grassland sites (uncropped sites with perennial vegetation and little or no external inputs). Land use and aridity were strong drivers of soil aggregation explaining 33% and 20% of the variation, respectively. SOC stocks were best explained by calcium content (20% of explained variation) followed by aridity (15%) and mean annual temperature (10%). We also found a threshold-like pattern for SOC stocks and aggregate stability in response to aridity, with lower values at sites with higher aridity. The impact of crop management on aggregate stability and SOC stocks appeared to be regulated by these thresholds, with more pronounced positive effects of crop diversity and more severe negative effects of crop management intensity in nondryland compared with dryland regions. We link the higher sensitivity of SOC stocks and aggregate stability in nondryland regions to a higher climatic potential for aggregate-mediated SOC stabilization. The presented findings are relevant for improving predictions of management effects on soil structure and C storage and highlight the need for site-specific agri-environmental policies to improve soil quality and C sequestration.

Agricultural management and pesticide use reduce the functioning of beneficial plant symbionts.

Phosphorus (P) acquisition is key for plant growth. Arbuscular mycorrhizal fungi (AMF) help plants acquire P from soil. Understanding which factors drive AMF-supported nutrient uptake is essential to develop more sustainable agroecosystems. Here we collected soils from 150 cereal fields and 60 non-cropped grassland sites across a 3,000 km trans-European gradient. In a greenhouse experiment, we tested the ability of AMF in these soils to forage for the radioisotope 33P from a hyphal compartment. AMF communities in grassland soils were much more efficient in acquiring 33P and transferred 64% more 33P to plants compared with AMF in cropland soils. Fungicide application best explained hyphal 33P transfer in cropland soils. The use of fungicides and subsequent decline in AMF richness in croplands reduced 33P uptake by 43%. Our results suggest that land-use intensity and fungicide use are major deterrents to the functioning and natural nutrient uptake capacity of AMF in agroecosystems.

Land-use intensification differentially affects bacterial, fungal and protist communities and decreases microbiome network complexity.

BACKGROUND: Soil microbial communities are major drivers of cycling of soil nutrients that sustain plant growth and productivity. Yet, a holistic understanding of the impact of land-use intensification on the soil microbiome is still poorly understood. Here, we used a field experiment to investigate the long-term consequences of changes in land-use intensity based on cropping frequency (continuous cropping, alternating cropping with a temporary grassland, perennial grassland) on bacterial, protist and fungal communities as well as on their co-occurrence networks. RESULTS: We showed that land use has a major impact on the structure and composition of bacterial, protist and fungal communities. Grassland and arable cropping differed markedly with many taxa differentiating between both land use types. The smallest differences in the microbiome were observed between temporary grassland and continuous cropping, which suggests lasting effects of the cropping system preceding the temporary grasslands. Land-use intensity also affected the bacterial co-occurrence networks with increased complexity in the perennial grassland comparing to the other land-use systems. Similarly, co-occurrence networks within microbial groups showed a higher connectivity in the perennial grasslands. Protists, particularly Rhizaria, dominated in soil microbial associations, as they showed a higher number of connections than bacteria and fungi in all land uses. CONCLUSIONS: Our findings provide evidence of legacy effects of prior land use on the composition of the soil microbiome. Whatever the land use, network analyses highlighted the importance of protists as a key element of the soil microbiome that should be considered in future work. Altogether, this work provides a holistic perspective of the differential responses of various microbial groups and of their associations to agricultural intensification.

Unraveling negative biotic interactions determining soil microbial community assembly and functioning.

Microbial communities play important roles in all ecosystems and yet a comprehensive understanding of the ecological processes governing the assembly of these communities is missing. To address the role of biotic interactions between microorganisms in assembly and for functioning of the soil microbiota, we used a top-down manipulation approach based on the removal of various populations in a natural soil microbial community. We hypothesized that removal of certain microbial groups will strongly affect the relative fitness of many others, therefore unraveling the contribution of biotic interactions in shaping the soil microbiome. Here we show that 39% of the dominant bacterial taxa across treatments were subjected to competitive interactions during soil recolonization, highlighting the importance of biotic interactions in the assembly of microbial communities in soil. Moreover, our approach allowed the identification of microbial community assembly rule as exemplified by the competitive exclusion between members of Bacillales and Proteobacteriales. Modified biotic interactions resulted in greater changes in activities related to N- than to C-cycling. Our approach can provide a new and promising avenue to study microbial interactions in complex ecosystems as well as the links between microbial community composition and ecosystem function.

Diversity of archaea and niche preferences among putative ammonia-oxidizing Nitrososphaeria dominating across European arable soils.

Archaeal communities in arable soils are dominated by Nitrososphaeria, a class within Thaumarchaeota comprising all known ammonia-oxidizing archaea (AOA). AOA are key players in the nitrogen cycle and defining their niche specialization can help predicting effects of environmental change on these communities. However, hierarchical effects of environmental filters on AOA and the delineation of niche preferences of nitrososphaerial lineages remain poorly understood. We used phylogenetic information at fine scale and machine learning approaches to identify climatic, edaphic and geomorphological drivers of Nitrososphaeria and other archaea along a 3000 km European gradient. Only limited insights into the ecology of the low-abundant archaeal classes could be inferred, but our analyses underlined the multifactorial nature of niche differentiation within Nitrososphaeria. Mean annual temperature, C:N ratio and pH were the best predictors of their diversity, evenness and distribution. Thresholds in the predictions could be defined for C:N ratio and cation exchange capacity. Furthermore, multiple, independent and recent specializations to soil pH were detected in the Nitrososphaeria phylogeny. The coexistence of widespread ecophysiological differences between closely related soil Nitrososphaeria highlights that their ecology is best studied at fine phylogenetic scale.

Crop cover is more important than rotational diversity for soil multifunctionality and cereal yields in European cropping systems.

In natural ecosystems, positive effects of plant diversity on ecosystem functioning have been widely observed, yet whether this is true in cropping systems remains unclear. Here we assessed the impact of crop diversification on soil microbial diversity, soil multifunctionality (SMF) and crop yields in 155 cereal fields across a 3,000 km north-south European gradient. Overall, crop diversity showed a relatively minor effect on soil microbial diversity, SMF and yields. In contrast, the proportion of time with crop cover (including cash crops, cover crops or forage leys) during the past ten-year crop rotation had a much stronger impact. This suggests that increasing crop cover can enhance both yields and soil functioning, while also providing habitat for soil microorganisms. We found that SMF did not positively contribute to crop yields, highlighting that care must be taken to balance the provision of food with environmentally beneficial functions and services, since they do not always go hand in hand.

Cover Crop Management Practices Rather Than Composition of Cover Crop Mixtures Affect Bacterial Communities in No-Till Agroecosystems.

Cover cropping plays a key role in the maintenance of arable soil health and the enhancement of agroecosystem services. However, our understanding of how cover crop management impacts soil microbial communities and how these interactions might affect soil nutrient cycling is still limited. Here, we studied the impact of four cover crop mixtures varying in species richness and functional diversity, three cover crop termination strategies (i.e., frost, rolling, and glyphosate) and two levels of irrigation at the cover crop sowing on soil nitrogen and carbon dynamics, soil microbial diversity, and structure as well as the abundance of total bacteria, archaea, and N-cycling microbial guilds. We found that total nitrogen and soil organic carbon were higher when cover crops were killed by frost compared to rolling and glyphosate termination treatments, while cover crop biomass was positively correlated to soil carbon and C:N ratio. Modifications of soil properties due to cover crop management rather than the composition of cover crop mixtures were related to changes in the abundance of ammonia oxidizers and denitrifiers, while there was no effect on the total bacterial abundance. Unraveling the underlying processes by which cover crop management shapes soil physico-chemical properties and bacterial communities is of importance to help selecting optimized agricultural practices for sustainable farming systems.

Impact of Leptospermone, a Natural ß-Triketone Herbicide, on the Fungal Composition and Diversity of Two Arable Soils.

Impact of leptospermone, a ß-triketone bioherbicide, was investigated on the fungal community which supports important soil ecological functions such as decomposition of organic matter and nutrients recycling. This study was done in a microcosm experiment using two French soils, Perpignan (P) and Saint-Jean-de-Fos (SJF), differing in their physicochemical properties and history treatment with synthetic ß-triketones. Soil microcosms were treated with leptospermone at recommended dose and incubated under controlled conditions for 45 days. Untreated microcosms were used as control. Illumina MiSeq sequencing of the internal transcribed spacer region of the fungal rRNA revealed significant changes in fungal community structure and diversity in both soils. Xylariales, Hypocreales, Pleosporales and Capnodiales (Ascomycota phyla) fungi and those belonging to Sebacinales, Cantharellales, Agaricales, Polyporales, Filobasidiales and Tremellales orders (Basidiomycota phyla) were well represented in treated soil microcosms compared to control. Nevertheless, while for the treated SJF a complete recovery of the fungal community was observed at the end of the experiment, this was not the case for the P treated soil, although no more bioherbicide remained. Indeed, the relative abundance of most of the saprophytic fungi were lower in treated soil compared to control microcosms whereas fungi from parasitic fungi included in Spizellomycetales and Pezizales orders increased. To the best of our knowledge, this is the only study assessing the effect of the bioherbicide leptospermone on the composition and diversity of the fungal community in soil. This study showed that leptospermone has an impact on α- and ß-diversity of the fungal community. It underlines the possible interest of microbial endpoints for environmental risk assessment of biopesticide.

Assessment of the ecotoxicological impact of natural and synthetic ß-triketone herbicides on the diversity and activity of the soil bacterial community using omic approaches.

The emergence of pesticides of natural origin appears as an environmental-friendly alternative to synthetic pesticides for managing weeds. To verify this assumption, leptospermone, a natural ß-triketone herbicide, and sulcotrione, a synthetic one, were applied to soil microcosms at 0× (control), 1× or 10× recommended field dose. The fate of these two herbicides (i.e. dissipation and formation of transformation products) was monitored to assess the scenario of exposure of soil microorganisms to natural and synthetic herbicides. Ecotoxicological impact of both herbicides was explored by monitoring soil bacterial diversity and activity using next-generation sequencing of 16S rRNA gene amplicons and soil metabolomics. Both leptospermone and sulcotrione fully dissipated over the incubation period. During their dissipation, transformation products of natural and synthetic ß-triketone were detected. Hydroxy-leptospermone was almost completely dissipated by the end of the experiment, while CMBA, the major metabolite of sulcotrione, remained in soil microcosms. After 8 days of exposure, the diversity and structure of the soil bacterial community treated with leptospermone was significantly modified, while less significant changes were observed for sulcotrione. For both herbicides, the diversity of the soil bacterial community was still not completely recovered by the end of the experiment (45 days). The combined use of next-generation sequencing and metabolomic approaches allowed us to assess the ecotoxicological impact of natural and synthetic pesticides on non-target soil microorganisms and to detect potential biomarkers of soil exposure to ß-triketones.

Evidence for photolytic and microbial degradation processes in the dissipation of leptospermone, a natural ß-triketone herbicide.

Bioherbicides appear as an ecofriendly alternative to synthetic herbicides, generally used for weed management, because they are supposed to have low side on human health and ecosystems. In this context, our work aims to study abiotic (i.e., photolysis) and biotic (i.e,. biodegradation) processes involved in the fate of leptospermone, a natural ß-triketone herbicide, by combining chemical and microbiological approaches. Under controlled conditions, the photolysis of leptospermone was sensitive to pH. Leptospermone has a half-life of 72 h under simulated solar light irradiations. Several transformation products, including hydroxy-leptospermone, were identified. For the first time, a bacterial strain able to degrade leptospermone was isolated from an arable soil. Based on its 16S ribosomal RNA (rRNA) gene sequence, it was affiliated to the Methylophilus group and was accordingly named as Methylophilus sp. LS1. Interestingly, we report that the abundance of OTUs, similar to the 16S rRNA gene sequence of Methylophilus sp. LS1, was strongly increased in soil treated with leptospermone. The leptospermone was completely dissipated by this bacteria, with a half-life time of 6 days, allowing concomitantly its growth. Hydroxy-leptospermone was identified in the bacterial culture as a major transformation product, allowing us to propose a pathway of transformation of leptospermone including both abiotic and biotic processes.

Ecotoxicological Impact of the Bioherbicide Leptospermone on the Microbial Community of Two Arable Soils.

The ecotoxicological impact of leptospermone, a ß-triketone bioherbicide, on the bacterial community of two arable soils was investigated. Soil microcosms were exposed to 0 × (control), 1 × or 10 × recommended dose of leptospermone. The ß-triketone was moderately adsorbed to both soils (i.e.,: K fa ~ 1.2 and K oc ~ 140 mL g(-1)). Its dissipation was lower in sterilized than in unsterilized soils suggesting that it was mainly influenced by biotic factors. Within 45 days, leptospermone disappeared almost entirely from one of the two soils (i.e., DT50 < 10 days), while 25% remained in the other. The composition of the microbial community assessed by qPCR targeting 11 microbial groups was found to be significantly modified in soil microcosms exposed to leptospermone. Pyrosequencing of 16S rRNA gene amplicons showed a shift in the bacterial community structure and a significant impact of leptospermone on the diversity of the soil bacterial community. Changes in the composition, and in the α- and ß-diversity of microbial community were transient in the soil able to fully dissipate the leptospermone, but were persistent in the soil where ß-triketone remained. To conclude the bacterial community of the two soils was sensitive to leptospermone and its resilience was observed only when leptospermone was fully dissipated.

Environmental Metabolic Footprinting: A novel application to study the impact of a natural and a synthetic ß-triketone herbicide in soil.

This study presents a novel approach for assessing the risk of agrochemicals in soil microcosms through the use of non-targeted metabolomics. The metabolome of treated soils was extracted and tested through LCMS profiling in order to generate an "Environmental Metabolic Footprint" (EMF). A dynamic characterization of pollution biomarkers was obtained through a multivariate statistical analysis of EMF data, where our results show the possible evolution towards a state of resilience. The EMF methodology was applied to two ß-triketone herbicides in soil microcosms: one natural, leptospermone, and one synthetic, sulcotrione. In spite of a four-fold higher application dose, leptospermone exhibited a lower resilience time than did sulcotrione (ca. 30 days vs ca. 45 days respectively).

The LPS O-Antigen in Photosynthetic Bradyrhizobium Strains Is Dispensable for the Establishment of a Successful Symbiosis with Aeschynomene Legumes.

The photosynthetic bradyrhizobia are able to use a Nod-factor independent process to induce nitrogen-fixing nodules on some semi-aquatic Aeschynomene species. These bacteria display a unique LPS O-antigen composed of a new sugar, the bradyrhizose that is regarded as a key symbiotic factor due to its non-immunogenic character. In this study, to check this hypothesis, we isolated mutants affected in the O-antigen synthesis by screening a transposon mutant library of the ORS285 strain for clones altered in colony morphology. Over the 10,000 mutants screened, five were selected and found to be mutated in two genes, rfaL, encoding for a putative O-antigen ligase and gdh encoding for a putative dTDP-glucose 4,6-dehydratase. Biochemical analysis confirmed that the LPS of these mutants completely lack the O-antigen region. However, no effect of the mutations could be detected on the symbiotic properties of the mutants indicating that the O-antigen region of photosynthetic Bradyrhizobium strains is not required for the establishment of symbiosis with Aeschynomene.

Isolation and characterization of Bradyrhizobium sp. SR1 degrading two ß-triketone herbicides.

In this study, a bacterial strain able to use sulcotrione, a ß-triketone herbicide, as sole source of carbon and energy was isolated from soil samples previously treated with this herbicide. Phylogenetic study based on16S rRNA gene sequence showed that the isolate has 100 % of similarity with several Bradyrhizobium and was accordingly designated as Bradyrhizobium sp. SR1. Plasmid profiling revealed the presence of a large plasmid (>50 kb) in SR1 not cured under nonselective conditions. Its transfer to Escherichia coli by electroporation failed to induce ß-triketone degrading capacity, suggesting that degrading genes possibly located on this plasmid cannot be expressed in E. coli or that they are not plasmid borne. The evaluation of the SR1 ability to degrade various synthetic (mesotrione and tembotrione) and natural (leptospermone) triketones showed that this strain was also able to degrade mesotrione. Although SR1 was able to entirely dissipate both herbicides, degradation rate of sulcotrione was ten times higher than that of mesotrione, showing a greater affinity of degrading-enzyme system to sulcotrione. Degradation pathway of sulcotrione involved the formation of 2-chloro-4-mesylbenzoic acid (CMBA), previously identified in sulcotrione degradation, and of a new metabolite identified as hydroxy-sulcotrione. Mesotrione degradation pathway leads to the accumulation of 4-methylsulfonyl-2-nitrobenzoic acid (MNBA) and 2-amino-4 methylsulfonylbenzoic acid (AMBA), two well-known metabolites of this herbicide. Along with the dissipation of ß-triketones, one could observe the decrease in 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibition, indicating that toxicity was due to parent molecules, and not to the formed metabolites. This is the first report of the isolation of bacterial strain able to transform two ß-triketones.