Coalescence events, which consist in the mixing of previously separated communities, are frequent in nature or as a result of human activities. Despite recently gaining attention as a tool to test ecological theories and engineer microbial communities, little is known about the factors that influence the outcome of such coalescence events. Here, we evaluated the relative importance of three community properties-namely, diversity, composition, and density-in determining coalescence outcome and biotic interactions among members of the coalescing bacterial communities. We found that manipulation of the density and composition of soil bacterial community resulted in the largest shifts in the structure of the resulting coalesced communities, explaining 24.7% and 6.8% of the variance in the β-diversity of the coalesced communities, respectively. Coalescence events impacted up to 35% of the dominant Operational Taxonomic Unit (OTUs) in the native community, with a predominance of negative effects. Our results also revealed that community density had the greatest explanatory power for the variance in the relative abundance of the OTUs negatively affected by coalescence events. In particular, all significantly affected OTUs that belonged to the Bacillales exhibited a decrease in relative abundance in several of the coalesced communities, which was related to the density of some members of the α-Proteobacteria and γ-Proteobacteria in the manipulated community suspensions. Overall, our data suggest that community density and composition were the main properties determining the outcome of coalescence events and that coalescence experiments can offer insights into multi-species interactions in complex environments.

Fungicides are used worldwide to improve crop yields, but they can affect non-target soil microorganisms which are essential for ecosystem functioning. Microorganisms form complex communities characterized by a myriad of interspecies interactions, yet it remains unclear to what extent non-target microorganisms are indirectly affected by fungicides through biotic interactions with sensitive taxa. To quantify such indirect effects, we fragmented a soil microbial community by filtration to alter biotic interactions and compared the effect of the fungicide hymexazol between fractions in soil microcosms. We postulated that OTUs which are indirectly affected would exhibit a different response to the fungicide across the fragmented communities. We found that hymexazol primarily affected bacterial and fungal communities through indirect effects, which were responsible for more than 75% of the shifts in relative abundance of the dominant microbial OTUs after exposure to an agronomic dose of hymexazol. However, these indirect effects decreased for the bacterial community when hymexazol doses increased. Our results also suggest that N-cycling processes such as ammonia oxidation can be impacted indirectly by fungicide application. This work sheds light on the indirect impact of fungicide exposure on soil microorganisms through biotic interactions, which underscores the need for higher-tier risk assessment.Environmental ImplicationIn this study, we used a novel approach based on the fragmentation of the soil microbial community to determine to which extent fungicide application could indirectly affect fungi and bacteria through biotic interactions. To assess off-target effects of fungicide on soil microorganisms, we selected hymexazol, which is used worldwide to control a variety of fungal plant pathogens, and exposed arable soil to the recommended field rate, as well as to higher rates. Our findings show that at least 75% of hymexazol-impacted microbial OTUs were indirectly affected, therefore emphasizing the importance of tiered risk assessment.

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.

Microbial inoculants are attracting growing interest in agriculture, but their efficacy remains unreliable in relation to their poor survival, partly due to the competition with the soil resident community. We hypothesised that recurrent inoculation could gradually alleviate this competition and improve the survival of the inoculant while increasing its impact on the resident bacterial community. We tested the effectiveness of such strategy with four inoculation sequences of Pseudomonas fluorescens strain B177 in soil microcosms with increasing number and frequency of inoculation, compared to a non-inoculated control. Each sequence was carried out at two inoculation densities (106 and 108 cfu.g soil−1). The four-inoculation sequence induced a higher abundance of P. fluorescens, 2 weeks after the last inoculation. No impact of inoculation sequences was observed on the resident community diversity and composition. Differential abundance analysis identified only 28 out of 576 dominants OTUs affected by the high-density inoculum, whatever the inoculation sequence. Recurrent inoculations induced a strong accumulation of nitrate, not explained by the abundance of nitrifying or nitrate-reducing microorganisms. In summary, inoculant density rather than inoculation pattern matters for inoculation effect on the resident bacterial communities, while recurrent inoculation allowed to slightly enhance the survival of the inoculant and strongly increased soil nitrate content.

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. We assessed the extent to which the soil fauna can shape microbial community assembly using an exclusion experiment in a grassland field to sort soil biota based on body size. Exclusion of the soil fauna according to body size led to cascading effects through the food web. In particular, exclusion of larger soil fauna positively impacted phagotrophic protists with subsequent effects on microbial community composition.image

Fungicides are an essential component of current agricultural practices, but their extensive use has raised concerns about their effects on non-target soil microorganisms, which carry out essential ecosystem functions. However, despite the complexity of microbial communities, many studies investigating their response to fungicides focus only on bacteria or fungi at one point in time. In this study, we used amplicon sequencing to assess the effect of the fungicide hymexazol on the diversity, composition, and co-occurrence network of soil bacteria, fungi, and protists at 7, 21, and 60 days after application. We found that hymexazol had very little effect on microbial alpha-diversity, but that microbial community composition and OTU differential abundance were altered over the duration of the experiment, even after hymexazol concentrations were undetectable. The co-occurrence patterns within and between microbial kingdoms were affected by hymexazol dose, suggesting that indirect effects may play a role in the microbial community response. Nitrogen cycling was also affected, with a transient hymexazol-associated increase in the abundance of ammonia-oxidizing microorganisms and soil nitrate concentration. These findings highlight that the effects of fungicides on soil microorganisms are dynamic and extensive, spanning several taxonomic kingdoms.

N2O emissions resulting from the application of nitrogen (N) fertilizers and vinasse represent the main sources of greenhouse gases (GHG) emissions in sugar-energy sector. Conversely, the application of biochar in soils has been worldwide recognized as an strategy to mitigate N2O emissions, although little is known about their effects on soils under energy cane production. The study aimed to evaluate the effects of biochar addition as a strategy to mitigate soil N2O emissions in soil under energy cane cultivation, as well as to quantify the abundance of N2O-producing and N2O-reducing microbial guilds. A greenhouse experiment was conducted in a completely randomized design, with five treatments and four replications. The treatments were: i) no N fertilization (control); ii) N fertilization; iii) N fertilization plus vinasse; iv) N fertilization plus biochar; v) N fertilization plus vinasse plus biochar. All treatments (except control) were balanced to receive the same amount of nutrients. Biochar was added at a rate of 5 g kg � 1 of soil. Soil N2O emissions were quantified by static chambers for 78 days, and soil sampling were performed to determine chemical and microbiological attributes, including functional genes of the nitrogen cycle (AOA, AOB, nirK, nirS, nosZI and nosZII) by real-time PCR. Results indicated that vinasse addition increased N2O emissions. Conversely, the application of biochar reduced N2O emissions associated with the application of N fertilizer (56 %) and N fertilizer + vinasse (41 %). The high N2O emissions observed in vinasse treatment were directly correlated with nitrifier microorganisms (AOB and AOA), indicating that nitrification should be the main pathway of N2O emissions in this treatment. The production of energy cane biomass was similar between N fertilizer treatments. High N2O emission intensities (mg N2O g biomass -1) were obtained in treatments with vinasse application. This study concluded that biochar is an effficient strategy to mitigate N2O emissions, providing the first insights into how biochar affects the microbial community associated with N2O emissions from soil under energy cane cultivation.

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.

Plant communities comprising species with different growth strategies and belonging to different functional groups can ensure stable productivity under variable climatic conditions. However, how plant communities can influence the response of nitrogen (N) cycling, in particular, soil microbial N cycling communities, N leaching and N2O fluxes under flooding, and their capacity to suppress flooding-induced N2O fluxes, remains unresolved. The aim of this study was to examine the effect of different plant communities composed of grasses and/or legumes on N cycling soil microorganisms and N2O fluxes, and how these effects are influenced by flooding. Our field experiment consisted of monocultures and two- and four-species mixtures of two grass and two legume species with different growth strategies (slow- and fast-growing species), grown in a fertilised sandy soil in the Netherlands. One year after plant establishment, we imposed paired control and flooding treatments for three weeks. We found that flooding significantly reduced plant N uptake and increased N2O fluxes. This increase was associated with higher abundances of N cycling microbial communities (except for ammonia-oxidising bacteria). Legume presence increased N2O fluxes, irrespective of the legume growth strategy or flooding, but this was not driven by changes in N cycling microbial communities; instead, it was related to an increase in soil nitrate availability. Mixing grasses with legumes promoted high plant N uptake and reduced N losses under control and flooded conditions, in particular when combining slow-growing species, and in the four-species mixture. Our results show that flooding exerted a strong influence on N cycling by increasing N leaching, N2O fluxes, microbial community abundances and decreasing plant N uptake. However, plant communities with slow-growing strategy had lowest relative abundance of nosZII bacteria and ameliorated flooding effects by both reducing N losses and enhancing plant N uptake.

Climate change models predict shifts in the frequency and magnitude of rain events (precipitation patterns). We studied how precipitation history shapes microbial community responses to rewetting and how these effects depend on N status. Twelve weeks of contrasting precipitation and N input left a legacy effect by shaping present (DNA-based) and potentially active (rRNA-based) bacterial and fungal communities. This legacy effect determined the microbial response to rewetting, as demonstrated by differences in the post-wet potentially active bacterial and fungal communities as well as the flux of soil-emitted CO2 over a 29-h period. Despite contrasting effects of precipitation and N input history on fungal:bacterial ratio and microbial community composition, the timing of the potentially active bacterial and fungal response to rewetting was not altered. Thus, regardless of precipitation or N input history, potentially active bacteria responded with a small shift in community composition within 1 h of rewetting but did not change further for the remaining 28 h analyzed. The potentially active fungi did not respond to rewetting within 29 h. Even though more extreme fluctuations in soil moisture changed soil microbial community composition, the short-term response of microbial communities to rewetting was conserved. Soil CO2 efflux upon rewetting was higher from systems with a history of frequent precipitation and underline the role of plants in drying-rewetting processes. We suggest that shifts in the fungal:bacterial ratio, as well as N-cycling potentials, may have consequences on food web stability and soil biogeochemical cycling.

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.

Abiotic factors have long been recognised as important factors in structuring microbial diversity and species associations, among which topography and hydro-geomorphic flows have an impact from plot to large scale. These factors are deeply involved in the dynamics of climate change. However, the actual impact of topography on microbial communities in spatially defined habitats remains unclear and, needing further development, represents a promising branch to investigate microbiological assets in the environment. In this study, we analysed a parcel in continental France which revealed a combined action of hydro-geomorphic fluxes and topography in structuring microbial assemblages. Species-habitat occurrence seems to respond to the effective energy locally displayed by fluxes. Largest richness and microbial variety occurred where fluxes are small such as on limited slope or reduced runoff concentrations. Species dominance was higher in zones with higher fluxes suggesting: 1) an impoverishment of the more sensible species, or 2) a selective adaptation of the most resistant species. This differentiation was evidenced by analysing the potential impact of topography and cumulated fluxes for runoff and sediments (i.e., WTI, LS RUSLE indexes) on microbial richness, dominance, and abondance at Phylum and Class levels.

Soil compaction caused by highly mechanized agriculture can constrain soil microbial diversity and functioning. Physical pressure on the soil decreases macropores and thereby limits oxygen diffusion. The associated shift from aerobic to anaerobic conditions can reduce nitrification and promote denitrification processes, leading to nitrogen (N) losses and N depletion that affect plant productivity. High soil moisture content during trafficking can exacerbate the negative effects of soil compaction. However, the extent to which soil moisture amplifies the effects of compaction on the soil microbiome and its control over N cycling is not well understood. Using a controlled greenhouse experiment with two different crops (pea and wheat), we compared the effects of compaction at three different soil moisture levels on soil physicochemical properties, microbial diversity, and the abundance of specific N species and quantification of associated microbial functional groups in the N cycle. Soil compaction increased bulk density from 15% (light compaction) to 25% (severe compaction). Compaction delayed germination in both crops and reduced yield by up to 60% for pea and 40% for wheat. Compaction further induced crop-specific shifts in microbial community structures. After compaction, the relative abundance of denitrifiers increased along with increased nitrate (NO3-) consumption and elevated nitrous oxide (N2O) concentrations in the soil pores. Conversely, the relative abundance of nitrifiers remained stable under compaction, but potentially decelerated nitrification rates, resulting in ammonium (NH4+) accumulation in the soil. This study showed that soil compaction effects are proportional to the initial soil moisture content, which could serve as a good indicator of compaction severity on agricultural fields. However, the impact of soil compaction on crop performance and on microbial communities and functions associated with the N cycle were not necessarily aligned. These findings demonstrate that not only the soil physical properties but also various biological indicators need to be considered in order to provide more precise recommendations for developing sustainable farming systems.

Artificial selection of microbiota opens new avenues for improving plants. However, reported results lack consistency. We hypothesised that the success in artificial selection of microbiota depends on the stabilisation of community structure. In a ten-generation experiment involving 1,800 plants, we selected rhizosphere microbiota of Brachypodium distachyon associated with high or low leaf greenness, a proxy of plant performance. The microbiota structure showed strong fluctuations during an initial transitory phase, with no detectable leaf greenness heritability. After five generations, the microbiota structure stabilised, concomitantly with heritability in leaf greenness. Selection, initially ineffective, did successfully alter the selected property as intended, especially for high selection. We show a remarkable correlation between the variability in plant traits and selected microbiota structures, revealing two distinct sub-communities associated with high or low leaf greenness, whose abundance was significantly steered by directional selection. Understanding microbiota structure stabilisation will improve the reliability of artificial microbiota selection.

The rhizosphere is a dynamic and complex interface between plant roots and microorganisms. Owing to exudates, a web of interactions establishes among the microbial members of this micro-environment. The present study explored the impact of a bacterial consortium (Azotobacter chroococcum, Bacillus megaterium and Pseudomonas fluorescens, ABP), on the fate of a human pathogen, Listeria monocytogenes EGD-e, in soil and in the rhizospheres of Cajanus cajan and Festuca arundinacea, in addition to its plant growth promoting effect. The study further assessed the impact these bioinoculants exert on the autochthonous soil bacterial communities. Experiments in sterilised soil inoculated with bioinoculants and L. monocytogenes revealed the inhibition of L. monocytogenes by approximately 80-fold compared to that without the consortium. Subsequently, experiments were conducted in non-sterile soil microcosms planted with C. cajan and F. arundinacea, and in bulk soil. The consortium led to a significant increase in plant growth in both plants and prevented growth of L. monocytogenes. However, the presence of resident soil bacterial communities overshadowed this inhibitory effect, and a sharp decline in L. monocytogenes populations (5–6 log reduction) was recorded under non-sterile soil conditions. A shift in the soil resident bacterial communities was observed upon amendment with the bioinoculants. A significant increase of potential Plant Growth Promoting Rhizobacteria (PGPR) and biocontrol agents was observed, while the abundance of potential phytopathogens dropped. The present study opens up new avenues for the application of such a consortium given their dual benefits of plant growth promotion and restricting phytopathogens as well as human pathogen.

Plant functional traits affect soil nutrient cycling in many ecosystems, but high levels of diversity make it challenging to identify their influence in tropical rainforests. Here, we used a litter manipulation experiment to demonstrate that differences in litter chemistry alone are sufficient to drive changes in soil nitrogen (N) cycling within a two-year period in a lowland tropical rainforest. Previously, we used airborne imaging spectroscopy to identify ten 0.25 ha plots on the Osa Peninsula of southwestern Costa Rica, each containing 10-20 emergent trees, with relatively high or low mean canopy N content (+/- 0.4 standard deviations of the landscape mean; n = 5 each). Plots with high canopy N content had higher soil inorganic N concentrations, faster net N cycling rates, and greater nitrous oxide (N2O) emissions. Because abiotic conditions were similar among the plot types, we hypothesized that litter inputs drove the formation of these high and low soil N patches. We tested this hypothesis with a reciprocal litter transplant experiment. After two years of monthly treatments, litter inputs from the high canopy N plots increased soil N availability in the low canopy N plots by 30% but had no measurable effects on net N cycling rates or N2O emissions. Surprisingly, litter inputs from the low canopy N plots increased soil N availability and net nitrification in the high canopy N plots. While somewhat counterintuitive from a canopy N perspective, these results may reflect a more nuanced effect of litter chemistry. Although low canopy N plot litter had lower N content than high canopy N plot litter, it leached relatively more and higher quality soluble carbon (C), which we speculate may have had a positive priming effect, releasing N from more recalcitrant compounds. Additionally, low canopy N plot litter supported higher rates of free-living N fixation, which could have directly increased soil N availability. Overall, our results show that tropical tree assemblages with differing foliar chemistry can directly influence soil properties through litter inputs, and that this effect may be mediated not only by litter N, but also relative C solubility and chemistry.

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.

Nitrification inhibitors have often been found to reduce nitrous oxide (N2O) emissions from agricultural soils, but N2O mitigation is variable and may depend on soil and climate variables. This study investigated how key abiotic factors modified the effect of Vizura (R), a new formulation with the nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) for livestock slurry, on N2O emissions. Two different incubations were carried out to investigate the main and interactive effects of selected factors on N2O emissions after manure application to soil. Experiment 1 studied two contrasting soil textures (coarse sandy vs sandy loam soil) and temperatures (10 vs 20 degrees C); Experiment 2 used the coarse sandy soil to compare two levels of soil pH (4.9 vs 6.4) and nitrate availability (0 vs 50 mg N kg(-1)). Nitrous oxide emissions and spatial distribution of mineral N were determined in both experiments, while temporal dynamics of mineral N pools and N-cycling microbial communities were investigated in Experiment 2 only. In sandy loam soil, the efficiency of DMPP to mitigate N2O emissions after manure application was similar at 10 and 20 degrees C, while in the coarse sandy soil DMPP was not effective at 10 degrees C, possibly because of low nitrification activity. Nitrous oxide emissions from the coarse sandy soil were higher at pH 4.9 than at pH 6.4, which was accompanied by lower abundance of nosZ genes and therefore potentially higher N2O/ (N-2+N2O) product ratios. DMPP significantly reduced the abundance of ammonia-oxidizing bacteria (AOB) on all sampling days, but had no effect on ammonia-oxidizing archaea (AOA). The inhibitor also reduced the abundance of comammox, which are newly discovered complete ammonia oxidizers. This study showed how soil conditions (soil texture, temperature, pH), through effects on nitrifiers and denitrifiers, and mineral N distribution, can modify the efficiency of nitrification inhibitors in reducing N2O emissions from livestock slurry.

Research on artificial selection of microbial community has become popular due to perspectives in improving plant and animal health 1-4 . However, reported results still lack consistency 5-8 . We hypothesized that artificial selection may provide desired outcomes provided that microbial community structure has stabilized along the selection process. In a ten-generation artificial selection experiment involving 1,800 plants, we selected rhizosphere microbiota of Brachypodium distachyon that were associated with high or low levels of leaf greenness, a proxy for plant health 9 . Monitoring of the rhizosphere microbiota dynamics showed strong oscillations in community structure during an initial transitory phase of five generations, with no heritability in the selected property. In the last five generations, the structure of microbial communities displayed signs of stabilization, concomitantly to the appearance of heritability in leaf greenness. Selection pressure, initially ineffective, became successful in changing the greenness index in the intended direction, especially toward high greenness values. We showed a remarkable congruence between plant traits and selected microbial community structures, highlighting two phylogenetically distinct microbial sub-communities correlating with leaf greenness, whose abundance was significantly steered by directional artificial selection. Understanding microbial community structure stabilization can thus help improve the reliability of artificial microbiota selection. Short Sentence Stable microbiota selection enables trait heritability