Bacterial wilt, caused by Ralstonia solanacearum (RS), is a major constraint on tomato production worldwide. This study investigated how multitrophic rhizosphere communities associated with five tomato cultivars (AM, DF, MF, NP, and ZY) respond to RS invasion using sterile plate and greenhouse pot assays. Rhizosphere bacterial and protistan communities were characterized by 16S and 18S rRNA gene amplicon sequencing under control and RS-inoculated conditions. Results showed that both cultivar identity and RS invasion significantly shaped microbial structures and compositions. RS infection exerted a stronger influence on bacterial communities than protists, whereas cultivar identity was the primary driver for protistan shifts. Random forest classification and differential abundance analysis identified specific microbial taxa that effectively discriminated cultivars and infection status. Community assembly analysis further revealed that bacterial assembly shifted from predominantly deterministic to more stochastic processes under pathogen pressure, while protistan assembly remained largely stochastic. Collectively, these results demonstrate that tomato cultivars harbor distinct multi-trophic rhizosphere microbial configurations under pathogen invasion, and highlight the importance of incorporating protists into rhizosphere community frameworks for understanding and potentially enhancing bacterial wilt suppression.

Although viruses are increasingly recognized as important contributors of soil carbon (C) cycling, their role in regulating soil C:N stoichiometry remains largely unclear. Here, we combined multi-omics approaches (viromics and metagenomics) with direct microcosm experiments to investigate viral contributions to soil C:N stoichiometry across three long-term fertilization field sites in China. Soils receiving long-term high N input exhibited distinct shifts in both taxonomic composition and functional gene profiles, with viruses showing a particularly strong contribution to C cycling. Mechanistically, soil viral and bacterial communities responded differentially to prolonged N enrichment, with viral communities exhibiting greater sensitivity to long-term N application. N addition reshaped the functional potential of both communities, with more pronounced changes in viral richness and virus-bacteria interactions. Elevated N levels enriched polyvalent viruses and increased the abundance of viral auxiliary metabolic genes involved in carbohydrate degradation. Direct experiments using viral transplants and metagenomic-stable isotope probing further confirmed that viruses can directly regulate nutrient cycling. Overall, our results demonstrate that soil viromes play a key role in regulating C cycling in ways that buffer Ninduced shifts in soil C:N ratios by reshaping the taxonomic and functional composition of soil microbial communities.

Many soil protists are bacterivores, yet how protist predation reshapes bacterial metabolic interactions and functions remains poorly understood. Here, we combine global soil samples with microbial metabolic simulations, along with soil microcosm-pot validations, to investigate the influence of protists on bacterial metabolic interactions. Across 3,785 metabolic simulations spanning 757 soils, increased protists predicted higher bacterial metabolic interaction potential and cross-feeding but lower metabolic resource overlap and competition. These patterns were confirmed using an independent rhizosphere dataset and metagenomic analysis. Protist predation selected bacterial communities containing GC-rich genomes, acid-carbon-preferring taxa, and enhanced metabolite exchange. Additionally, exposing a synthetic community (SynCom) to protist predation elevated the expression of bacterial genes associated with plant growth-promoting functions. Consistently, microcosm- and pot-based experiments showed that protist addition increased bacterial cross-feeding over time and improved plant performance. Together, we establish a scalable framework to evaluate protist-driven bacterial cooperation and function to guide rational rhizosphere microbiome engineering.

Composting organic waste is a sustainable recycling method in agricultural systems, yet the microbial preferences for different substrates and their influence on composting efficiency remain underexplored. Here, 210 datasets of published 16S ribosomal DNA amplicon sequences from straw and manure composts worldwide were analyzed, and a database of 278 bacterial isolates was compiled. Substrate-driven microbiome variations were most prominent during the initial composting stages. Indigenous synthetic communities exhibit substrate-specific adaptations, increasing compost temperatures by 2 %-10 %, microbial abundance by 44 %-233 %, and microbial activity by 26 %-60 %. Key dissolved substrates, such as choline and succinic acid in straw compost, and phloretin and uric acid in manure compost, drive these microbial preferences. These findings highlight how substrate-specific microbiomes can be engineered to enhance microbial activity, accelerate temperature rise, and extend the thermophilic phase, providing a targeted framework to improve composting efficiency and tailor strategies to different organic waste types.

Plant growth-promoting rhizobacteria(PGPR)have been widely used for the promotion of plant performance.Predatory protists can influence the taxonomic and functional composition of rhizosphere bacteria.However,research on the impact of the interaction between protist and PGPR on plant performance remains at a very early stage.Here,we examined the impacts of individual inoculation of protist(Colpoda inflata,Dimastigella trypaniformis,or Vermamoeba vermiformis)or the PGPR strain Bacillus velezensis SQR9 as well as the co-inoculation of the protist C.inflata and B.velezensis SQR9 on the growth of tomato plants.We found that all individual protists and Bacillus could promote plant growth compared to the control with no microbe inoculation,with the co-inoculation of C.inflata and B.velezensis SQR9 achieving the greatest performance,including plant height,fresh weight,and dry weight.Different protists harbored distinct rhizosphere bacterial communities,with the co-inoculation of protist and Bacillus resulting in the lowest bacterial diversity and driving significant changes in community structure and composition,particularly by increasing the relative abundance of Proteobacteria.Random forest model highlighted Cellvibrio as the most important bacterial predictor of plant growth,which was enriched after protist inoculation,especially after the mixed inoculation of protist and Bacillus.We further found that bacterial functional genes of nitrogen metabolism were the key determinants of plant growth.These results indicate that the interaction between protists and Bacillus can support plant growth by reshaping rhizosphere bacterial community composition and function.Understanding the interaction mechanisms between protist and PGPR is crucial for their effective utilization in sustainable agriculture.

Soil protists play vital roles in influencing plant performance, yet their interactions with plant-beneficial bacteria are still poorly understood. Here, we examine how two soil protists (Naegleria sp. and Cercomonas sp.) affect the pathogen Ralstonia solanacearum, both on individual beneficial bacteria and within a synthetic microbial community (SynCom). Combining in vitro and pot experiments, we find that the SynCom together with Naegleria provided significantly greater suppression of the pathogen (enhanced suppressiveness by 74.29% compared to SynCom alone). Additionally, Naegleria increases SynCom biofilm biomass by 2.44 times. Population dynamics tracking revealed that Naegleria enriched Bacillus populations, leading to a positive correlation between Bacillus and Pseudomonas. Metatranscriptomics analysis shows upregulation of genes related to biofilm formation (such as epsA-O and tapA-sipW-tasA operon) and secondary metabolite biosynthesis (e.g., macrolactin H, bacillaene, and difficidin) in the presence of Naegleria. Our study demonstrates that Naegleria enhances plant health by predating on pathogens, promoting beneficial bacteria, and stimulating protective microbial functions in the rhizosphere.

Phyllosphere microbiota play crucial roles in supporting host performance. However, the dynamic changes of phyllosphere-associated microbiome during pathogen infections and their impacts on plant health remain unknown. Here, we found phyllosphere microbes can mitigate wheat Fusarium head blight (FHB), a severe disease caused by Fusarium graminearum (F. graminearum) pathogen that promotes infection by inducing host alkalinization. Using wheat head microbial community profiling and metatranscriptomics, we found Pseudomonas spp. significantly enriched on infected wheat heads. Through isolating 595 bacterial strains from infected wheat heads-including 196 Pseudomonas isolates-we identified certain enriched Pseudomonas isolates capable of producing organic acids that counteract pathogen-induced pH upshift. In vitro experiments confirm the selective promotion of specific host-acidifying Pseudomonas in wheat heads. Field trials confirmed that host-acidifying Pseudomonas strains effectively controlled FHB. These findings highlight the pivotal role of plant-beneficial microbes in host pH regulation and offer innovative avenues for sustainable plant disease control.

Fusarium wilt is increasingly threatening banana production around the world. Investigating soil microbial communities associated with healthy and diseased banana plants is the first step to understand the potential mechanisms involved in the disease suppression. Previous research has confirmed plant-beneficial bacterial and fungal communities are key determinants of banana health. However, to what extent protists, a key component of the soil microbiome, are linked to banana health on a large scale remains largely unknown. Here, we collected soil samples from healthy and diseased plants suffering from Fusarium wilt in multiple banana plantations within China and Laos, and examined holistic soil microbial communities including bacteria, fungi and protists using high-throughput sequencing. We explored the linkage between protists and Fusarium oxysporum and investigated the effects of biotic and abiotic factors on protists. Results showed the relative abundance of Fusarium oxysporum can be highly predicted by protists. Specifically, predatory protists revealed a negative correlation with F. oxysporum, which was confirmed in pot experiments. We found the putative plant growth-promoting bacteria, positively correlated with predatory protists, were also negatively correlated with F. oxysporum. In addition, both soil abiotic factors (i.e., soil pH and ammonia nitrogen) and biotic factors (soil bacteria) played crucial roles in determining predatory protists. We highlighted that soil predatory protists might contribute to banana health via directly inhibiting soil-borne pathogens or indirectly enriching plant beneficial bacteria.

Hyperthermophilic composting (HTC) is a promising strategy for the treatment of organic solid waste, leveraging extreme thermophilic conditions (up to 90°C) driven by specialized microbial communities. While microbial community composition and succession have been previously described during HTC, the metabolic activity and adaptation of thermophilic microbiomes remain largely unexplored. In this study, we conducted time-series metagenomic and metatranscriptomic analyses on samples from a full-scale HTC system to characterize the composition, functional potential, and metabolic activity of thermophilic bacteria. A total of 227 non-redundant metagenome-assembled genomes (MAGs) were recovered, including 45 thermophilic MAGs (optimal growth temperatures > 45°C). Metatranscriptomic profiling revealed that thermophilic taxa-such as Thermus thermophilus, Planifilum fulgidum, and Thermaerobacter spp.-were highly transcriptionally active and played vital roles in heat generation through the upregulation of energy production and carbohydrate metabolism pathways. Additionally, these thermophiles exhibited survival and adaptation strategies involving physiological changes (e.g., spore formation, enhanced motility, and genome streamlining) and the induction of thermal resistance mechanisms (e.g., DNA repair systems, heat-shock proteins, and synthesis of compatible solutes). Overall, this study provides novel insights into the diverse survival strategies of thermophilic microbiomes in HTC and suggests potential avenues for optimizing thermophilic biotreatment processes for solid waste management. IMPORTANCE:Despite increasing interest in hyperthermophilic composting as a sustainable waste treatment strategy, the mechanisms by which microbial communities both tolerate and drive extreme thermal conditions remain unclear. This study fills a critical knowledge gap by identifying a small group of highly active thermophilic bacteria that dominate during peak composting temperatures and orchestrate endogenous heat production. Using genome-resolved multi-omics, we demonstrate that these thermophiles couple high metabolic output with specialized survival strategies-such as genome streamlining, thermotolerance systems, and adaptive motility systems. These findings advance our understanding of microbial function under extreme conditions and provide a framework for optimizing thermophilic microbiome performance in engineered ecosystems.

Crop domestication has long been known to reshape rhizosphere microbial communities, yet research has focused disproprotionately on bacteria and fungal responses to crop domestication while neglecting protist communities. Protists, as key microbial predators regulating bacterial populations and thereby their functionalities, remain understudied in this context. Here, we investigate the influence of soybean domestication on both bacterial and protist communities, with a focus on the reorganization of ecological strategies, specifically generalists and specialists, within these microbiomes. We analyzed 270 rhizosphere samples from 27 domesticated and 63 wild soybean varieties. Domestication significantly altered community compositions of bacterial communities, with wild soybeans harboring higher proprotions of Pseudomonadota (71.4 %) and Bacillota (4.8 %), while domesticated soybeans exhibited an enrichment of Bacteroidota (11.0 %). Protist communities also diverged: wild soybeans were dominated by Cercozoa (58.2 %) and Gyrista (23.5 %), while domesticated plants had more Ciliophora (7.1 %) and Evosea (5.7 %). Domesticated soybeans hosted fewer generalist and specialist bacteria but more generalist protists, suggesting divergent microbial responses to domestication. Correlation analyses revealed that bacterial and protist generalists exhibited strong positive correlations with each other. At the same time, bacterial and protist specialists also showed positive correlations in wild soybeans-patterns that were largely absent in their domesticated counterparts. Functionally, wild soybeans supported more ureolytic and methylotrophic bacteria, while domesticated soybeans favored nitrate-respiration taxa. Notably, predatory protists in wild soybeans were significantly correlated with bacteria involved in carbon and nitrogen cycling, a key ecological relationship lost with domestication. These findings suggest that domestication exerts different selection pressures on bacteria and protists, disrupting potential relationships between bacterial and protist functional groups.

Antimicrobial resistance poses a substantial and growing threat to global health. While antibiotic resistance genes (ARGs) are tracked most closely in clinical settings, their spread remains poorly understood in non-clinical environments. Mitigating the spread of ARGs in non-clinical contexts such as soil could limit their enrichment in food webs. Multi-omics (involving metagenomics, metatranscriptomics, viromics, and metabolomics) and direct experimentation show that targeting keystone bacterial taxa by phages can limit ARG maintenance and dissemination in natural soil environments. Based on the metagenomic analysis, we first show that phages from activated sludge can regulate soil microbiome composition and function in terms of reducing ARG abundances and changing the bacterial community composition. This effect was mainly driven by a reduction in the abundance and activity of Streptomyces genus, which is well known for encoding both antibiotic resistance and synthesis genes. To validate the significance of this keystone species for the loss of ARGs, we enriched phage consortia specific to Streptomyces and tested their effect on ARG abundances on 48 soil samples collected across China. We observed a consistent reduction in ARG abundances across all soils, confirming that Streptomyces-enriched phages could predictably change the soil microbiome resistome and mitigate the prevalence of ARGs. This study highlights that phages can be used as ecosystem engineers to control the spread of antibiotic resistance in the environment. Our study demonstrates that some bacterial keystone taxa are critical for ARG maintenance and dissemination in soil microbiomes, and opens new ecological avenues for microbiome modification and resistome control. This study advances our understanding of how metagenomics-informed phage consortia can be used to predictably regulate soil microbiome composition and functioning by targeting keystone bacterial taxa.

The COVID-19 pandemic has profoundly impacted the environment due to changes in human activities, including the widespread use of disinfectants. While previous studies have shown that increased disinfectant use during the pandemic affected the antibiotic resistome in aquatic environments, its impact on soils remains largely unexplored. In this study, we analyzed 332 soil metagenomic samples collected across China before (2017-2019) and during (2020-2022) the pandemic to assess the effects on antibiotic resistance genes (ARGs), mobile genetic elements (MGEs), and the soil bacterial community. Our results revealed a significant increase in the abundance of ARGs and MGEs in farmland soils during the COVID-19 pandemic. Moreover, the abundance of potentially pathogenic bacteria, such as Pseudomonas, Salmonella, and Vibrio, increased significantly during the pandemic. Partial least squares path modeling indicated that disinfectant use was a key factor associated with the spread of ARGs, primarily by enhancing MGE-mediated gene transfer. These findings suggest that shifts in human activity during the pandemic have contributed to the dissemination of soil ARGs and underscore the importance of managing microbial resistance within the One Health framework.

Viruses are the most abundant microbial entities on Earth, playing a critical role in elemental cycling. However, to date, there is no experimental evidence demonstrating whether viruses participate in nitrogen (N) cycling in soil. Here, we combined stable isotope probing (SIP) and metagenomics to detect 15N assimilation by viruses and their putative bacterial hosts in soil microcosms incubated with 15N-labeled N2. We recovered 609 viral operational taxonomic units (vOTUs, > 5 kb) and 49 metagenome–assembled genomes (MAGs) from the 15N-labeled soils using metagenomics. Based on metagenomic–SIP, a total of 65 vOTUs and 10 MAGs with potential N–transforming abilities were identified due to their exclusive enrichment in the heavy fractions under 15N2 treatment compared to 14N2, indicating their significance for soil N transformation. Moreover, three N–fixing MAGs (active diazotrophs) and one lytic virus with the potential to infect these diazotrophs were observed in the 15N-labeled soil. This indicates that viruses can assimilate 15N into their DNA via infection of diazotrophs. Additionally, two auxiliary metabolic genes associated with N cycling were identified in two viruses, suggesting that viruses may provision their hosts with N-cycling genes. Overall, our results demonstrate that soil viruses can promote microbial N turnover through viral lysis, highlighting the viral shunt as an important mechanism facilitating elemental cycling in soils.

Arbuscular mycorrhizal fungi (AMF) represent a crucial component of soil microorganisms, playing pivotal roles in promoting plant growth by enhancing nutrient availability. However, the responses of AMF communities to different fertilization regimes and their correlations with plant communities in the context of anthropogenic disturbances in alpine meadow ecosystems remain largely unexplored. In this study, we investigated the effects of nitrogen, phosphorus, and combined nitrogen-phosphorus fertilization on AMF communities and their interconnections with plant diversity and biomass based on a seven-year long-term experiment conducted on the Qinghai-Tibet Plateau. Our results showed significant shifts in AMF community structure and composition under different fertilization treatments, while the richness of AMF exhibited no remarkable alterations. Notably, soil pH decreased, and electrical conductivity increased with the increasing nitrogen fertilizer application, emerging as pivotal abiotic factors in predicting plant richness and biomass. Fascinatingly, Acaulospora exhibited a positive correlation with plant richness, serving as an important bioindicator of plant richness, while Diversispora emerged as the primary bioindicator of plant biomass. Our findings shed light on potential correlations between AMF community composition and both plant and soil abiotic factors, driven by nitrogen and phosphorus fertilization. We advocate for the critical significance of balanced fertilization in sustaining beneficial plant–soil–AMF interactions in natural ecosystems as well as agricultural soils.

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

ABSTRACT While the distribution of extracellular ARGs (eARGs) in the environment has been widely reported, the factors governing their release remain poorly understood. Here, we combined multi-omics and direct experimentation to test whether the release and transmission of eARGs are associated with viral lysis and heat during cow manure composting. Our results reveal that the proportion of eARGs increased 2.7-fold during composting, despite a significant and concomitant reduction in intracellular ARG abundances. This relative increase of eARGs was driven by composting temperature and viral lysis of ARG-carrying bacteria based on metagenome-assembled genome (MAG) analysis. Notably, thermal lysis of mesophilic bacteria carrying ARGs was a key factor in releasing eARGs at the thermophilic phase, while viral lysis played a relatively stronger role during the non-thermal phase of composting. Furthermore, MAG-based tracking of ARGs in combination with direct transformation experiments demonstrated that eARGs released during composting pose a potential transmission risk. Our study provides bioinformatic and experimental evidence of the undiscovered role of temperature and viral lysis in co-driving the spread of ARGs in compost microbiomes via the horizontal transfer of environmentally released DNA. IMPORTANCE The spread of antibiotic resistance genes (ARGs) is a critical global health concern. Understanding the factors influencing the release of extracellular ARGs (eARGs) is essential for developing effective strategies. In this study, we investigated the association between viral lysis, heat, and eARG release during composting. Our findings revealed a substantial increase in eARGs despite reduced intracellular ARG abundance. Composting temperature and viral lysis were identified as key drivers, with thermal lysis predominant during the thermophilic phase and viral lysis during non-thermal phases. Moreover, eARGs released during composting posed a transmission risk through horizontal gene transfer. This study highlights the significance of temperature and phage lysis in ARG spread, providing valuable insights for mitigating antibiotic resistance threats.

Antibiotic resistance has grown into a major public health threat. In this study, we reveal predation by protists as an overlooked driver of antibiotic resistance dissemination in the soil microbiome. While previous studies have primarily focused on the distribution of antibiotic resistance genes, our work sheds light on the pivotal role of soil protists in shaping antibiotic resistance dynamics. Using a combination of metagenomics and controlled experiments in this study, we demonstrate that protists cause an increase in antibiotic resistance. We mechanistically link this increase to a fostering of antimicrobial activity in the microbiome. Protist predation gives a competitive edge to bacteria capable of producing antagonistic secondary metabolites, which secondary metabolites promote in turn antibiotic-resistant bacteria. This study provides insights into the complex interplay between protists and soil microbiomes in regulating antibiotic resistance dynamics. This study highlights the importance of top-down control on the spread of antibiotic resistance and directly connects it to cross-kingdom interactions within the microbiome. Managing protist communities may become an important tool to control outbreaks of antibiotic resistance in the environment.

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

While beneficial plant-microbe interactions are common in nature, direct evidence for the evolution of bacterial mutualism is scarce. Here we use experimental evolution to causally show that initially plant-antagonistic Pseudomonas protegens bacteria evolve into mutualists in the rhizosphere of Arabidopsis thaliana within six plant growth cycles (6 months). This evolutionary transition is accompanied with increased mutualist fitness via two mechanisms: (i) improved competitiveness for root exudates and (ii) enhanced tolerance to the plant-secreted antimicrobial scopoletin whose production is regulated by transcription factor MYB72 . Crucially, these mutualistic adaptations are coupled with reduced phytotoxicity, enhanced transcription of MYB72 in roots, and a positive effect on plant growth. Genetically, mutualism is associated with diverse mutations in the GacS/GacA two-component regulator system, which confers high fitness benefits only in the presence of plants. Together, our results show that rhizosphere bacteria can rapidly evolve along the parasitism-mutualism continuum at an agriculturally relevant evolutionary timescale.

Gentiana atropurpurea is an annual herb belonging to section Microsperma T.N. Ho series Suborbisepalae Marquand. This species is endemic to China with its distribution limited to the southeast of the QTP. In this study, the complete chloroplast genome sequence of G. atropurpurea was characterized from Illumina pair-end sequencing. The chloroplast genome of G. atropurpurea was 145,757 bp in length, containing a large single-copy region (LSC) of 78,287 bp, a small single-copy region (SSC) of 16,750 bp, and two inverted repeat (IR) regions of 25,360 bp. The overall GC content is 37.90%, while the corresponding values of the LSC, SSC, and IR regions are 35.8, 31.7, and 43.4%, respectively. The genome contains 132 complete genes, including 86 protein-coding genes (62 protein-coding gene species), 37 tRNA genes (29 tRNA species), and eight rRNA genes (four rRNA species). Phylogenetic analysis based on complete chloroplast genomes showed that G. atropurpurea and G. tongolensis clustered together as sisters to other related species.