Engineering natural microbiomes toward enhanced bioremediation by microbiome modeling.

Engineering natural microbiomes for biotechnological applications remains challenging, as metabolic interactions within microbiomes are largely unknown, and practical principles and tools for microbiome engineering are still lacking. Here, we present a combinatory top-down and bottom-up framework to engineer natural microbiomes for the construction of function-enhanced synthetic microbiomes. We show that application of herbicide and herbicide-degrader inoculation drives a convergent succession of different natural microbiomes toward functional microbiomes (e.g., enhanced bioremediation of herbicide-contaminated soils). We develop a metabolic modeling pipeline, SuperCC, that can be used to document metabolic interactions within microbiomes and to simulate the performances of different microbiomes. Using SuperCC, we construct bioremediation-enhanced synthetic microbiomes based on 18 keystone species identified from natural microbiomes. Our results highlight the importance of metabolic interactions in shaping microbiome functions and provide practical guidance for engineering natural microbiomes.

Exploiting microbial competition to promote plant health.

The host-associated microbiota can promote colonization resistance against pathogens via a mechanism termed 'nutrient blocking', as highlighted in a recent article by Spragge et al. This implies that greater metabolic overlap between commensal taxa and pathogens leads to disease suppression. Here, we discuss future avenues for how this principle can be exploited in the rhizosphere microbiota to promote plant health.

Fertilization Weakens the Ecological Succession of Dissolved Organic Matter in Paddy Rice Rhizosphere Soil at the Molecular Level.

Dissolved organic matter (DOM) is involved in numerous biogeochemical processes, and understanding the ecological succession of DOM is crucial for predicting its response to farming (e.g., fertilization) practices. Although plentiful studies have examined how fertilization practice affects the content of soil DOM, it remains unknown how long-term fertilization drives the succession of soil DOM over temporal scales. Here, we investigated the succession of DOM in paddy rice rhizosphere soils subjected to different long-term fertilization treatments (CK: no fertilization; NPK: inorganic fertilization; OM: organic fertilization) along with plant growth. Our results demonstrated that long-term fertilization significantly promoted the molecular chemodiversity of DOM, but it weakened the correlation between DOM composition and plant development. Time-decay analysis indicated that the DOM composition had a shorter halving time under CK treatment (94.7 days), compared to NPK (337.4 days) and OM (223.8 days) treatments, reflecting a lower molecular turnover rate of DOM under fertilization. Moreover, plant development significantly affected the assembly process of DOM only under CK, not under NPK and OM treatments. Taken together, our results demonstrated that long-term fertilization, especially inorganic fertilization, greatly weakens the ecological succession of DOM in the plant rhizosphere, which has a profound implication for understanding the complex plant-DOM interactions.

Fossil-fuel-dependent scenarios could lead to a significant decline of global plant-beneficial bacteria abundance in soils by 2100.

Exploiting the potential benefits of plant-associated microbes represents a sustainable approach to enhancing crop productivity. Plant-beneficial bacteria (PBB) provide multiple benefits to plants. However, the biogeography and community structure remain largely unknown. Here we constructed a PBB database to couple microbial taxonomy with their plant-beneficial traits and analysed the global atlas of potential PBB from 4,245 soil samples. We show that the diversity of PBB peaks in low-latitude regions, following a strong latitudinal diversity gradient. The distribution of potential PBB was primarily governed by environmental filtering, which was mainly determined by local climate. Our projections showed that fossil-fuel-dependent future scenarios would lead to a significant decline of potential PBB by 2100, especially biocontrol agents (-1.03%) and stress resistance bacteria (-0.61%), which may potentially threaten global food production and (agro)ecosystem services.

Global diversity and biogeography of potential phytopathogenic fungi in a changing world.

Phytopathogenic fungi threaten global food security but the ecological drivers of their global diversity and biogeography remain unknown. Here, we construct and analyse a global atlas of potential phytopathogenic fungi from 20,312 samples across all continents and major oceanic island regions, eleven land cover types, and twelve habitat types. We show a peak in the diversity of phytopathogenic fungi in mid-latitude regions, in contrast to the latitudinal diversity gradients observed in aboveground organisms. Our study identifies climate as an important driver of the global distribution of phytopathogenic fungi, and our models suggest that their diversity and invasion potential will increase globally by 2100. Importantly, phytopathogen diversity will increase largely in forest (37.27-79.12%) and cropland (34.93-82.51%) ecosystems, and this becomes more pronounced under fossil-fuelled industry dependent future scenarios. Thus, we recommend improved biomonitoring in forests and croplands, and optimised sustainable development approaches to reduce potential threats from phytopathogenic fungi.

Changes in soil stoichiometry, soil organic carbon mineralization and bacterial community assembly processes across soil profiles.

Soil organic carbon (SOC) mineralization is essential to biogeochemical recycling in terrestrial ecosystem. However, the microbial mechanisms underlying the nutrient-induced SOC mineralization remain uncertain. Here, we investigated how SOC mineralization was linked to microbial assembly processes as well as soil nutrient availability and stoichiometric ratio in a paddy rice ecosystem at four soil profile levels. Our results showed a sharp decrease in SOC mineralization from topsoil (112.61-146.34 mg CO2 kg-1 day-1) to subsoil (33.51-61.41 mg CO2 kg-1 day-1). High-throughput sequencing showed that both abundance and diversity of specialist microorganisms (Chao1: 1244.30-1341.35) significantly increased along the soil profile, while the generalist microorganisms (Chao1: 427.67-616.15; Shannon: 7.46-7.97) showed the opposite trend. Correspondingly, the proportion of deterministic processes that regulate specialist (9.64-21.59 %) and generalist microorganisms (21.17-53.53 %) increased and decreased from topsoil to subsoil, respectively. Linear regression modeling and partial least squares path modeling indicated that SOC mineralization was primarily controlled by the assembly processes of specialist microorganisms, which was significantly mediated by available soil C:N:P stoichiometry. This study highlighted the importance of soil stoichiometry-mediated bacterial community assembly processes in regulating SOC mineralization. Our results have an important implication for the integration of bacterial community assembly processes into the prediction of SOC dynamics.

Diseased-induced multifaceted variations in community assembly and functions of plant-associated microbiomes.

Plant-associated microorganisms are believed to be part of the so-called extended plant phenotypes, affecting plant growth and health. Understanding how plant-associated microorganisms respond to pathogen invasion is crucial to controlling plant diseases through microbiome manipulation. In this study, healthy and diseased (bacterial wilt disease, BWD) tomato (Solanum lycopersicum L.) plants were harvested, and variations in the rhizosphere and root endosphere microbial communities were subsequently investigated using amplicon and shotgun metagenome sequencing. BWD led to a significant increase in rhizosphere bacterial diversity in the rhizosphere but reduced bacterial diversity in the root endosphere. The ecological null model indicated that BWD enhanced the bacterial deterministic processes in both the rhizosphere and root endosphere. Network analysis showed that microbial co-occurrence complexity was increased in BWD-infected plants. Moreover, higher universal ecological dynamics of microbial communities were observed in the diseased rhizosphere. Metagenomic analysis revealed the enrichment of more functional gene pathways in the infected rhizosphere. More importantly, when tomato plants were infected with BWD, some plant-harmful pathways such as quorum sensing were significantly enriched, while some plant-beneficial pathways such as streptomycin biosynthesis were depleted. These findings broaden the understanding of plant-microbiome interactions and provide new clues to the underlying mechanism behind the interaction between the plant microbiome and BWD.

Supercapacitive performance of C-axis preferentially oriented TiO2 nanotube arrays decorated with MoO3 nanoparticles.

Highly ordered TiO2 nanotube arrays (TNTAs) have received great attention owing to their high surface area, stability and direct transport pathways. The TNTAs, modified with other materials exhibiting enhanced conductivity and capacitance have been considered to be promising anode materials for supercapacitors. In this work, MoO3/carbon@different crystallography-oriented TiO2 nanotube arrays (CTNTAs) were synthesized by an anodizing method and electrochemical deposition. The structure and morphology of the samples were characterized by X-ray diffraction (XRD), field emission scanning electron microscopy (FE-SEM), high-resolution transmission electron microscopy (HR-TEM) and X-ray photoelectron spectroscopy (XPS). The electrochemical performance was tested by cyclic voltammogram (CV) and galvanostatic charge-discharge (GDC) tests. The results indicated that MoO3/carbon@(004) preferentially oriented TiO2 nanotube array electrodes have the advantages of combining p-TNTAs and MoO3 nanoparticles and exhibit high electrochemical performance and cycling stability. The highest specific capacitance of the MoO3-p-CTNTA electrode achieved is 194 F g-1 at a current density of 1 A g-1.

Sustained detoxification of 1,2-dichloroethane to ethylene by a symbiotic consortium containing Dehalococcoides species.

1,2-Dichloroethane (1,2-DCA) is a ubiquitous volatile halogenated organic pollutant in groundwater and soil, which poses a serious threat to the ecosystem and human health. Microbial reductive dechlorination has been recognized as an environmentally-friendly strategy for the remediation of sites contaminated with 1,2-DCA. In this study, we obtained an anaerobic microbiota derived from 1,2-DCA contaminated groundwater, which was able to sustainably convert 1,2-DCA into non-toxic ethylene with an average dechlorination rate of 30.70 ± 11.06 µM d-1 (N = 6). The microbial community profile demonstrated that the relative abundance of Dehalococcoides species increased from 0.53 ± 0.08% to 44.68 ± 3.61% in parallel with the dechlorination of 1,2-DCA. Quantitative PCR results showed that the Dehalococcoides species 16S rRNA gene increased from 2.40 ± 1.71 × 108 copies∙mL-1 culture to 4.07 ± 2.45 × 108 copies∙mL-1 culture after dechlorinating 110.69 ± 30.61 µmol of 1,2-DCA with a growth yield of 1.55 ± 0.93 × 108 cells per µmol Cl- released (N = 6), suggesting that Dehalococcoides species used 1,2-DCA for organohalide respiration to maintain cell growth. Notably, the relative abundances of Methanobacterium sp. (p = 0.0618) and Desulfovibrio sp. (p = 0.0001995) also increased significantly during the dechlorination of 1,2-DCA and were clustered in the same module with Dehalococcoides species in the co-occurrence network. These results hinted that Dehalococcoides species, the obligate organohalide-respiring bacterium, exhibited potential symbiotic relationships with Methanobacterium and Desulfovibrio species. This study illustrates the importance of microbial interactions within functional microbiota and provides a promising microbial resource for in situ bioremediation in sites contaminated with 1,2-DCA.

Integrating pH into the metabolic theory of ecology to predict bacterial diversity in soil.

Microorganisms play essential roles in soil ecosystem functioning and maintenance, but methods are currently lacking for quantitative assessments of the mechanisms underlying microbial diversity patterns observed across disparate systems and scales. Here we established a quantitative model to incorporate pH into metabolic theory to capture and explain some of the unexplained variation in the relationship between temperature and soil bacterial diversity. We then tested and validated our newly developed models across multiple scales of ecological organization. At the species level, we modeled the diversification rate of the model bacterium Pseudomonas fluorescens evolving under laboratory media gradients varying in temperature and pH. At the community level, we modeled patterns of bacterial communities in paddy soils across a continental scale, which included natural gradients of pH and temperature. Last, we further extended our model at a global scale by integrating a meta-analysis comprising 870 soils collected worldwide from a wide range of ecosystems. Our results were robust in consistently predicting the distributional patterns of bacterial diversity across soil temperature and pH gradients-with model variation explaining from 7 to 66% of the variation in bacterial diversity, depending on the scale and system complexity. Together, our study represents a nexus point for the integration of soil bacterial diversity and quantitative models with the potential to be used at distinct spatiotemporal scales. By mechanistically representing pH into metabolic theory, our study enhances our capacity to explain and predict the patterns of bacterial diversity and functioning under current or future climate change scenarios.

Integrative analysis of microbiome and metabolome reveals the linkage between gut microbiota and carp growth.

Gut microbiota and their metabolites are increasingly recognized for their crucial role in regulating the health and growth of the host. The mechanism by which the gut microbiome affects the growth rate of fish (Cyprinus carpio) in the rice-fish coculture system, however, remains unclear. In this study, the gut contents of the fast-growing and slow-growing (FG and SG) carp were collected from the rice-fish coculture system for both the fish gut microbiome and metabolome analyses. High throughput 16 S rRNA gene sequencing showed that the overall gut microbiota of FG group was distinct from that of SG group. For example, the cyanobacteria were highly enriched in the guts of SG carp (18.61%), in contrast, they only represented a minor fraction of gut microbiota for FG group (<0.20%). The liquid chromatography-mass spectrometry (LC-MS)-based metabolomics analysis revealed that 191 identified metabolites mostly located in 18 KEGG pathways were differentially present between the two groups, of which more than 50% of these metabolites were involved in lipid and amino acids metabolism. Compared with the FG group, the gut microbiota of SG group significantly enriched the metabolic pathways involved in the steroid (hormone) biosynthesis, whereas reducing those associated with beta-alanine metabolism, biosynthesis of unsaturated fatty acids and bile secretion. The enrichment and depletion of these metabolic pathways resulted in an increase in steroid metabolites and a decrease in the concentration of spermidine, which may have a major impact on the growth rate of carp. The metabolome results were further supported by the predicated KEGG functions of the gut microbiomes of the two groups, pointing out that the gut microbiota could substantially affect the growth of fish via their unique metabolic functions. Together, our integrated fish gut microbiome and metabolome analysis has substantial implications for the development of engineered microbiome technologies in aquaculture.

Electron acceptors determine the BTEX degradation capacity of anaerobic microbiota via regulating the microbial community.

Anaerobic degradation is the major pathway for microbial degradation of benzene, toluene, ethylbenzene, and xylenes (BTEX) under electron acceptor lacking conditions. However, how exogenous electron acceptors modulate BTEX degradation through shaping the microbial community structure remains poorly understood. Here, we investigated the effect of various exogenous electron acceptors on BTEX degradation as well as methane production in anaerobic microbiota, which were enriched from the same contaminated soil. It was found that the BTEX degradation capacities of the anaerobic microbiota gradually increased along with the increasing redox potentials of the exogenous electron acceptors supplemented (WE: Without exogenous electron acceptors < SS: Sulfate supplement < FS: Ferric iron supplement < NS: Nitrate supplement), while the complexity of the co-occurring networks (e.g., avgK and links) of the microbiota gradually decreased, showing that microbiota supplemented with higher redox potential electron acceptors were less dependent on the formation of complex microbial interactions to perform BTEX degradation. Microbiota NS showed the highest degrading capacity and the broadest substrate-spectrum for BTEX, and it could metabolize BTEX through multiple modules which not only contained fewer species but also different key microbial taxa (eg. Petrimonas, Achromobacter and Comamonas). Microbiota WE and FS, with the highest methanogenic capacities, shared common core species such as Sedimentibacter, Acetobacterium, Methanobacterium and Smithella/Syntrophus, which cooperated with Geobacter (microbiota WE) or Desulfoprunum (microbiota FS) to perform BTEX degradation and methane production. This study demonstrates that electron acceptors may alter microbial function by reshaping microbial community structure and regulating microbial interactions and provides guidelines for electron acceptor selection for bioremediation of aromatic pollutant-contaminated anaerobic sites.

Using Potential Molecular Transformation To Understand the Molecular Trade-Offs in Soil Dissolved Organic Matter.

Understanding the chemical composition and molecular transformation in soil dissolved organic matter (DOM) is important to the global carbon cycle. To address this issue, ultrahigh-resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) was applied to investigate DOM molecules in 36 paddy soils collected from subtropical China. All the detected 7576 unique molecules were divided into seven compound groups, and nine trade-off relationships between different compound groups were revealed based on principal component analysis and Pearson's correlation. An optimized method was developed to evaluate all potential molecular transformations in DOM samples. The concept of thermodynamics was introduced to evaluate the identified molecular transformations and classify them as thermodynamically favorable (TFP) and thermodynamically limited (TLP) processes. Here, we first tried to understand the molecular trade-offs by using the potential molecular transformations. All the nine trade-offs could be explained by molecular transformations. Six trade-offs had bases of biochemical reactions, and the trade-off-related direct transformations could explain the content variations of carbohydrate-like, condensed aromatic-like, tannin-like, and lignin-like compounds in TLP. More reasonable explanations existed in the TLP rather than TFP, which demonstrated the critical role of external energy in the molecular transformation of soil DOM.

Reduced chemodiversity suppresses rhizosphere microbiome functioning in the mono-cropped agroecosystems.

BACKGROUND: Rhizodeposits regulate rhizosphere interactions, processes, nutrient and energy flow, and plant-microbe communication and thus play a vital role in maintaining soil and plant health. However, it remains unclear whether and how alteration in belowground carbon allocation and chemodiversity of rhizodeposits influences microbiome functioning in the rhizosphere ecosystems. To address this research gap, we investigated the relationship of rhizosphere carbon allocation and chemodiversity with microbiome biodiversity and functioning during peanut (Arachis hypogaea) continuous mono-cropping. After continuously labeling plants with 13CO2, we studied the chemodiversity and composition of rhizodeposits, along with the composition and diversity of active rhizosphere microbiome using metabolomic, amplicon, and shotgun metagenomic sequencing approaches based on DNA stable-isotope probing (DNA-SIP). RESULTS: Our results indicated that enrichment and depletion of rhizodeposits and active microbial taxa varied across plant growth stages and cropping durations. Specifically, a gradual decrease in the rhizosphere carbon allocation, chemodiversity, biodiversity and abundance of plant-beneficial taxa (such as Gemmatimonas, Streptomyces, Ramlibacter, and Lysobacter), and functional gene pathways (such as quorum sensing and biosynthesis of antibiotics) was observed with years of mono-cropping. We detected significant and strong correlations between rhizodeposits and rhizosphere microbiome biodiversity and functioning, though these were regulated by different ecological processes. For instance, rhizodeposits and active bacterial communities were mainly governed by deterministic and stochastic processes, respectively. Overall, the reduction in carbon deposition and chemodiversity during peanut continuous mono-cropping tended to suppress microbial biodiversity and its functions in the rhizosphere ecosystem. CONCLUSIONS: Our results, for the first time, provide the evidence underlying the mechanism of rhizosphere microbiome malfunctioning in mono-cropped systems. Our study opens new avenues to deeply disentangle the complex plant-microbe interactions from the perspective of rhizodeposits chemodiversity and composition and will serve to guide future microbiome research for improving the functioning and services of soil ecosystems. Video abstract.

Phosphorus availability increases pathobiome abundance and invasion of rhizosphere microbial networks by Ralstonia.

Soil disease-suppressiveness depends on complex interactions among pathogens, native microbiota, and physicochemical properties, while these interactions remain understudied. Comparing field and microcosm experiments, we investigated the significance of these interactions in disease emergence or suppression using structural equation modelling (SEM) and receiver operating characteristic curve (ROC) analyses. We observed significant differences in the relative abundance of pathogenic and beneficial microbes, alpha and beta diversity indices between disease-conducive and -suppressive rhizosphere soils. The pathogenic (Ralstonia) and beneficial (Bacillus) taxa dominated disease-conducive and -suppressive rhizosphere soils, respectively. Moreover, the co-occurrences of Ralstonia with native microorganisms were positive and negative in the disease-conducive and -suppressive soils, respectively. These results suggest the supportive (Rudaea) and suppressive (Enterobacter, Bacillus) role of indigenous microbes in the invasion of soil and plant systems by Ralstonia. The SEM and ROC analysis predicted that Ralstonia invaded rhizospheric microbial networks and caused peanut wilt under high than low soil phosphorus conditions. Our results suggest the importance of soil phosphorus availability in altering the microbial interactions, thus leading to soil invasion by Ralstonia. Thus, we conclude by saying that feeding soil with high amounts of available phosphorus could deplete plant-beneficial microbes and increase the pathobiome abundance that may compromise plant health.

Assessing the ecological risk of pesticides should not ignore the impact of their transformation byproducts - The case of chlorantraniliprole.

Risk assessments for pesticides typically focus on the compound itself ignoring the impact of its transformation byproducts. Challenges in isolating such byproducts (i.e. after application of pesticide in soil) often lead to underestimation of the real risk from such substances. The toxicological properties of these byproducts may differ from those of the parent pesticides; hence, special attention is required for these new emerging contaminants. In this study, two transformation byproducts of chlorantraniliprole were isolated from soil and identified, using nuclear magnetic resonance and high resolution mass spectrometry, as products of dechlorination (Z1) and bromination (Z2). Kinetic experiments revealed both byproducts degrade faster than chlorantraniliprole in soil (half-lives 38 & 43 d vs. 58 d). The ecological risk evaluation of chlorantraniliprole and its byproducts on soil bacterial community showed that they were all potentially harmful but they imposed different impacts on both alpha and beta diversities and co-occurrence networks of the bacterial community. Z2 had the biggest potential impact on soil bacteria and accounted as a high potential risk. By comparing their impacts on soil bacterial community, we confirm that ecological risk assessment necessitates the understanding of the environmental impacts of a substance as well as of its transformation byproducts.

Variations in bacterial taxonomic profiles and potential functions in response to the gut transit of earthworms (Eisenia fetida) feeding on cow manure.

Earthworms play an important role in the organic matter decomposition in terrestrial ecosystems. Earthworms interact directly with the microorganisms to affect the organic matter decomposition via gut transit, i.e., the digestion and assimilation of organic matter in the foregut and midgut and its excretion by the hindgut. However, how the microbial community ingested by earthworms respond to the transit processes in different gut segments of earthworms is not clear. We used composted cow manure to feed earthworms and sampled vermicompost and the contents of foregut, midgut and hindgut for bacterial 16S rRNA gene sequencing analysis. We observed that earthworm gut transit decreased the abundances of the dominant phyla Proteobacteria and Bacteroidetes but increased Actinobacteria, Chloroflexi and Acidobacteria. The alpha diversity of bacterial community in midgut was the lowest of the different gut segments, and the bacterial community structure of the foregut was significantly different from the midgut and hindgut. The enrichment analysis results revealed different selective stimulatory and inhibitory effects on the ingested bacterial community in the different gut segments, which extended to vermicompost. The FAPROTAX data indicated that C and N metabolic microbes were enriched in the earthworm gut. Microbes involved in fermentation and methanogenesis were enriched in the hindgut, and denitrification microbes were enriched in the foregut. The N metabolism microbes in vermicompost were significantly enriched after the stimulation of earthworm gut transit (P < 0.05), and the pathogenic microbes of animals and plants were inhibited. Combined with the results of subsequent correlation and biochemical analyses, earthworm gut transit significantly altered the structure and function of the bacterial community to accelerate the degradation and mineralization of organic matter and the enrichment of phosphorus and potassium. Our study suggests that the gut transit process of earthworms plays an important role in regulating organic matter dynamics in terrestrial ecosystems.

Molecular level changes during suppression of Rhizoctonia solani growth by humic substances and relationships with chemical structure.

Elucidation of the inhibitory effects of humic substances (HSs) on phytopathogenic fungi and the underlying molecular mechanisms are highly important for improved biocontrol. In this study, we investigated the growth suppression, morphological characteristics, transcriptomic sequence, and radical signals of Rhizoctonia solani following HS addition (50 mg/L). Through mycelial cultured experiment, mycelia growth of R. solani had been suppressed with HS addition, and the inhibition rate was 24.88 ± 0.11% compared to the control. Field emission-scanning electron microscopy showed increased and superimposed branching mycelial growth, with a shriveled appearance. RNA samples of R. solani cultured with or without HSs were both extracted to examine the sequence on molecular level by Illumina HiSeq sequencing platform. RNA sequencing analysis revealed 175 differentially expressed genes (DEGs; 111 upregulated and 64 downregulated) between the HSs treatment and control. The upregulated unigenes were annotated and significantly enriched to three molecular processes: vitamin B6 metabolism, ABC transporters, and glutathione metabolism, while the downregulated unigenes were annotated to carbohydrate metabolism, but not significantly enriched. Real time-quantitative polymerase chain reaction analysis showed that the unigenes related to hexokinase, glucose-6-phosphate isomerase, glutathione synthase, and glutathione reductase were significantly decreased (by 60.03%, 70.70%, 60.33%, and 57.59%, respectively), while those related to glutathione S-transferase were significantly increased (2.66-fold). The electron paramagnetic resonance spectra showed that HSs induced increased the intensity of radical signals of R. solani in a cultured system increased by 59.56% compared to CK (without HSs addition). Network analysis based on DEGs expression and the chemical structure of HSs revealed that the carbonyl moiety in HSs formed the most links with nodes of the DEGs (sum of the links of positive and negative effects = 70), implicating this structure as the active fraction responsible for the inhibitory effect. This study provides molecular and chemical evidence of the biofungicidal activity of HSs with the potential for practical application.

Spatial Variation in Soil Fungal Communities across Paddy Fields in Subtropical China.

Fungi underpin almost all terrestrial ecosystem functions, yet our understanding of their community ecology lags far behind that of other organisms. Here, red paddy soils in subtropical China were collected across a soil depth profile, comprising 0-to-10-cm- (0-10cm-), 10-20cm-, and 20-40cm-deep layers. Using Illumina MiSeq amplicon sequencing of the internal transcribed spacer (ITS) region, distance-decay relationships (DDRs), and ecological models, fungal assemblages and their spatial patterns were investigated from each soil depth. We observed significant spatial variation in fungal communities and found that environmental heterogeneity decreased with soil depth, while spatial variation in fungal communities showed the opposite trend. DDRs occurred only in 0-10cm- and 10-20cm-deep soil layers, not in the 20-40cm layer. Our analyses revealed that the fungal community assembly in the 0-10cm layer was primarily governed by environmental filtering and a high dispersal rate, while in the deeper layer (20-40cm), it was primarily governed by dispersal limitation with minimal environmental filtering. Both environmental filtering and dispersal limitation controlled fungal community assembly in the 10-20cm layer, with dispersal limitation playing the major role. Results demonstrate the decreasing importance of environmental filtering and an increase in the importance of dispersal limitation in structuring fungal communities from shallower to deeper soils. Effectively, "everything is everywhere, but the environment selects," although only in shallower soils that are easily accessible to dispersive fungal propagules. This work highlights that perceived drivers of fungal community assembly are dependent on sampling depth, suggesting that caution is required when interpreting diversity patterns from samples that integrate across depths.IMPORTANCE In this work, Illumina MiSeq amplicon sequencing of the ITS region was used to investigate the spatial variation and assembly mechanisms of fungal communities from different soil layers across paddy fields in subtropical China, and the results demonstrate the decreasing importance of environmental filtering and an increase in the importance of dispersal limitation in structuring fungal communities from shallower to deeper soils. Therefore, the results of this study highlight that perceived drivers of fungal community assembly are dependent on sampling depth and suggest that caution is required when interpreting diversity patterns from samples that integrate across depths. This is the first study focusing on assemblages of fungal communities in different soil layers on a relatively large scale, and we thus believe that this study is of great importance to researchers and readers in microbial ecology, especially in microbial biogeography, because the results can provide sampling guidance in future studies of microbial biogeography.