The Nectriaceae includes major plant and human pathogens, yet the genomic foundation underpinning its taxonomy remains uneven and largely unassessed. We analysed 1,530 genome sequence assemblies to quantify metadata completeness, geographic and taxonomic bias, and assembly quality across the family. One-third of the assemblies lacked essential metadata, sequencing was heavily skewed toward a few agriculturally important lineages, and sampling of many genera was limited or nonexistent. BUSCO and QUAST metrics revealed striking heterogeneity in assembly quality, with widespread fragmentation and a substantial subset of genomes falling outside the expected quality thresholds. From orthologous protein sequences of 763 single-copy genes in 576 high-quality genomes, we reconstructed a phylogenomic backbone for the Nectriaceae and quantified gene- and site-level concordance. While major clades broadly match current concepts, extensive gene-tree discordance and a polyphyletic Nisikadoi complex highlight unresolved evolutionary and taxonomic boundaries. Our study delivers the first integrated, family-wide evaluation of Nectriaceae genomic resources and outlines a framework for quality standards, curated metadata, and stable phylogenomic inference to support future taxonomic and comparative work. ### Competing Interest Statement The authors have declared no competing interest.
Reliable evolutionary inference increasingly depends on public genome resources, and the effects of uneven assembly quality, incomplete metadata, and biased taxonomic sampling remain poorly quantified. Using the species-rich fungal lineage Nectriaceae as a model system, we analysed 1530 genome sequence assemblies to assess metadata completeness, sampling representation, and genome quality. One-third of the assemblies lacked essential metadata, sequencing was heavily skewed toward a few agriculturally important lineages, and sampling of many genera was limited or nonexistent. BUSCO and QUAST metrics revealed substantial heterogeneity in assembly quality, with widespread fragmentation and numerous assemblies falling outside expected quality thresholds. From 763 single-copy orthologs identified in 576 higher-quality genomes, we reconstructed a phylogenomic backbone and quantified gene- and site-level concordance across the tree. Although major clades were broadly recovered, extensive gene-tree discordance and a polyphyletic Fusarium nisikadoi species complex revealed unresolved boundaries and conflict among loci. These results show how data quality, incomplete sampling, and discordant genomic histories can constrain phylogenomic resolution, and provide a general framework for improving comparative genomic resources and large-scale evolutionary inference.
Arsenic contamination in paddy soils threatens crop productivity and results in the accumulation of toxic arsenic in rice grains. Here, we demonstrate that a defined synthetic microbial community (SynCom) of Priestia flexa and Pseudomonas putida mitigates arsenic toxicity and restricts arsenic accumulation in rice (Oryza sativa var. Sarju-52). Under arsenic stress [As(III), 18 mg/kg; As(V), 50 mg/kg], plants exhibited impaired growth, reduced photosynthetic performance, and increased oxidative stress. Inoculation with SynCom restored physiological function and metabolic balance, as evidenced by improved photosynthesis, increased soluble sugars, and reduced proline accumulation. Accompanied by attenuation of antioxidant enzyme overactivation, which indicates effective control of reactive oxygen species. Mechanistically, SynCom substantially reduced arsenic accumulation in roots, shoots and grains by coordinate downregulation of arsenic transporter genes (Lsi1, Lsi2, Lsi3, OsNIP1;1, and OsNIP3;3), which limits arsenic uptake and translocation. Metabolomic profiling also indicated stress-associated metabolite suppression and enrichment of growth-related pathways. These results demonstrate that microbiome engineering can reprogram plant responses to arsenic stress and provide a scalable strategy to reduce dietary arsenic exposure from staple crops. Additionally, this study lays a strong foundation for developing SynCom as an effective and sustainable biotechnological intervention to improve food safety and agricultural resilience in arsenic-contaminated areas.
Trichoderma spp. produce a diverse repertoire of metabolites with specific activities that contribute to biocontrol through direct antagonism, ecological signalling, and modulation of plant responses. However, current knowledge remains uneven: many metabolites are chemically described, whereas fewer are supported by robust gene-metabolite associations, experimentally validated ecological functions, and realistic translational evidence. Progress in this field will depend less on expanding compound catalogues than on integrating mechanistic, ecological, and translational evidence. This review examines the specialized metabolism of Trichoderma with emphasis on biosynthetic gene clusters, regulatory networks, ecological roles, and biosafety constraints relevant to biocontrol. Major metabolite classes, including polyketides, terpenoids, peptaibols, siderophores, diketopiperazines, and volatile organic compounds, are discussed together with representative case studies for which genetic and functional evidence is available. We further propose a translational framework to distinguish metabolites with mainly descriptive support from those approaching application readiness, based on four criteria: gene-level validation, demonstrated ecological role, manageable biosafety profile, and feasible delivery/stability. This perspective helps explain why metabolite inventories continue to expand faster than field translation. Recent advances in genomics, transcriptomics, metabolomics, genome editing, and formulation science are reshaping how Trichoderma metabolites are prioritized for future development.
The intestinal tract is a complex ecological interface in which host cells and resident microbiota jointly maintain barrier integrity, immune homeostasis, and metabolic balance. Within this ecosystem, pathogenic bacteria must overcome colonization resistance imposed by commensal communities and host defenses to establish infection. The type VI secretion system (T6SS) is a contact-dependent secretion apparatus that delivers effector proteins into bacterial competitors or host cells, thereby contributing to interbacterial competition, microbiota remodeling, and host-pathogen interactions. However, T6SS activity is not constitutive; rather, it is tightly regulated in response to environmental and host-derived cues. Two-component systems (TCSs) are central to this regulation because they sense intestinal signals, including pH variation, nutrient/metal ion availability and host-associated signals, and can convert these inputs into coordinated control of T6SS expression and activity, including, in some systems, assembly, effector deployment, and attack timing. In this review, we discuss how pathogens deploy TCS‑regulated T6SSs to orchestrate offensive antagonism and defensive countermeasures during intestinal infection. Beyond promoting T6SS-dependent antagonism, some TCSs also participate in damage sensing, envelope repair, counterattack responses, and compensatory defense programs. In certain T6SS-deficient contexts, TCS-mediated induction of capsular polysaccharide synthesis may provide an alternative protective strategy against T6SS-mediated killing, highlighting the regulatory plasticity of bacterial defense systems. By integrating current knowledge from signal perception to effector delivery, this review outlines how TCSs fine-tune bacterial competitiveness and resilience in the intestinal microenvironment and identifies key gaps that limit our understanding of TCS-T6SS regulation in vivo.
Nitrate impairs both symbiotic and free-living biological nitrogen fixation (BNF). While nitrate-induced phosphorylation signalling has been implicated in the inhibition of symbiotic BNF, the suppression of free-living BNF has generally been attributed to ammonium generated during nitrate assimilation. However, whether nitrate can inhibit free-living BNF independently of ammonium feedback regulation remains unclear. Here, an ammonium-deregulated mutant of Azotobacter chroococcum (A4) was used to investigate whether nitrate inhibits nitrogen fixation independently of ammonium regulation. Despite the loss of ammonium-mediated inhibition, nitrate significantly suppressed nitrogen fixation. Nitrate at concentrations above 2 mM reduced extracellular ammonium accumulation, with 10 mM nitrate decreasing ammonium production to 61% of that observed under nitrogen-free conditions. Integrated multi-omics analyses revealed that nitrate triggered extensive regulatory reprogramming across multiple molecular layers, with both coordinated and layer-specific responses across transcriptomic, proteomic and phosphoproteomic levels. These responses differed from the typical ammonium-mediated feedback regulation characterized by substantial repression of nitrogen fixation-related genes or proteins, but nevertheless resulted in reduced ammonium excretion, accompanied by enhanced biomass accumulation and extracellular polymeric substance (EPS) production in A4. Together, these findings indicate that, rather than directly repressing the nitrogen fixation machinery, prolonged nitrate exposure suppresses nitrogen fixation output through global regulatory reprogramming that redirects cellular metabolism and resource allocation away from nitrogen fixation. This study provides new insights into nitrate-mediated regulation of free-living diazotrophs and has implications for optimising nitrogen management and improving the application of nitrogen-fixing microorganisms.
Verticillium dahliae is a destructive soil-borne fungus with a broad host range, and its persistence in soil complicates control. Current measures, mainly resistant cultivars and chemicals, are limited and environmentally risky, promoting biocontrol as a green alternative. Here, we investigated the biocontrol mechanisms of Bacillus velezensis L33a against V. dahliae JR2 in tomato. In vitro assays on PDA plates at 26°C for 9 d showed that L33a inhibited JR2 by 58.6%, caused hyphal malformation and disruption, and its volatile organic compounds suppressed pathogen growth. In pot experiments, tomato roots dipped in JR2 suspension (1 ×10⁶ CFU/mL) for 30 min at 7 d after transplanting and grown for 21 d achieved 60.9% control efficacy. Physiological assays indicated reduced peroxidase and catalase activities, while qPCR revealed that L33a alone upregulated JA signaling (SlJAZ1, SlMYC2, SlPI II) and antioxidant (SlCAT, SlAPX) genes, with further enhancement upon JR2 co-treatment. To track their interactions, we generated GFP-labeled JR2 and RFP-labeled L33a; dual fluorescence labeling showed that L33a endophytically colonized Arabidopsis thaliana roots and competed with JR2 for the same niche, correlating with reduced pathogen colonization. Integrated metabolomic and transcriptomic analysis further revealed that L33a treatment altered pathways related to ABC transporters, amino acid metabolism, cell wall integrity, and energy metabolism in JR2, with tyrosine metabolism significantly enriched at both levels. Collectively, these findings suggest that L33a is a promising biocontrol strain for green management of tomato Verticillium wilt.
Salinity is a major abiotic stress that severely restricts crop productivity. Despite considerable potential, the role of Trichoderma afroharzianum T22 in the molecular responses and root microbiome dynamics associated with salinity tolerance remains poorly understood in sorghum. In this study, T. afroharzianum inoculation alleviated salinity-induced stress by improving chlorophyll content, growth parameters, and nutrient balance, while restricting root-to-shoot Na+ translocation. Split-root experiments showed that T. afroharzianum application to a single root compartment was insufficient to improve whole-plant performance under salinity, whereas inoculation of both compartments restored growth and chlorophyll-related traits. RNA-seq analysis showed the upregulation of genes involved in symbiosis, hormone signaling, antioxidant defense, and ion homeostasis, accompanied by repression of genes involved in ethylene biosynthesis and senescence in the roots. KEGG enrichment analysis further revealed activation of secondary metabolic pathways involved in stress adaptation. Furthermore, 16S rRNA sequencing showed that T. afroharzianum inoculation was associated with shifts in the root bacterial community without significantly altering alpha diversity, while selectively enriching putatively beneficial taxa, including Dyella mobilis, Luteibacter rhizovicinus, and Luteibacter yeojuensis under salinity. In addition, a conserved core microbiome was retained across treatments and was dominated by Streptomyces, Rhizobium, Dyella, and Labrys. Further, Janibacter was identified as a characteristic indicator taxon of T. afroharzianum inoculation, while Streptomyces showed the highest overall indicator value. Multi-omics integration analysis revealed that T. afroharzianum-associated microbial taxa were strongly associated with hormone signaling, redox homeostasis, mineral transport, and secondary metabolism under salinity stress. Particularly, Streptomyces and Luteibacter were the two genera most strongly associated with plant growth traits, whereas Rhizobium and Mucilaginibacter showed stronger positive correlations with tissue Na+ accumulation. Collectively, these findings provide new insights into T. afroharzianum-mediated salinity tolerance in sorghum and highlight its potential as a microbial biostimulant, warranting further validation across diverse sorghum genotypes in field conditions.
Cyclic di-GMP (c-di-GMP) is a universal bacterial second messenger that orchestrates the transition between motile and sessile lifestyles, thereby shaping microbial physiology, virulence, and persistence. In Escherichia coli (E. coli), c-di-GMP functions as a central integrator of environmental cues, dynamically regulating motility, biofilm formation, energy metabolism, and pathogenicity. Here, we provide a comprehensive synthesis of recent advances in c-di-GMP signaling in E. coli, spanning local signaling modules that control curli and cellulose biosynthesis to global regulatory circuits linking central metabolism, stress adaptation, and virulence gene expression. We further highlight the emerging view of c-di-GMP as a pathogen-associated molecular pattern that interfaces with host immune sensors such as STING and DDX41, extending its biological relevance beyond bacterial physiology. Finally, we discuss the translational potential of targeting c-di-GMP signaling for biofilm disruption, enhancement of bacteriophage-mediated clearance, and vaccine adjuvant design. Collectively, this review positions E. coli as a powerful model to decode the multifaceted biology of c-di-GMP and to inspire novel antimicrobial and immunomodulatory strategies.
Staphylococcus aureus (S. aureus)-induced mastitis represents a significant challenge in dairy production. Oyster peptides (OP), bioactive compounds derived from oysters, exhibit considerable therapeutic value in treating various diseases due to their antibacterial and anti-inflammatory properties. Nevertheless, their role in alleviating mastitis and protecting the blood-milk barrier (BMB) from S. aureus infection remains underexplored. This study reveals that OP effectively attenuates S. aureus-induced inflammation, reduces oxidative stress, and maintains BMB integrity by preserving tight junction protein expression. Mechanistic studies indicate that OP suppresses the S. aureus-induced over-activation of the TLR2-NF-κB and IP3R1/GRP75/VDAC1 signaling pathways, thereby mitigating inflammation and mitochondrial damage. In addition, 16S rDNA sequencing of the gut microbiota demonstrates that OP modulates microbial composition, enhancing the abundance of beneficial bacteria such as Bacteroides acidifaciens while downregulating harmful bacterial populations. Metabolomic analysis also indicates that OP affects intestinal metabolic pathways, increasing the levels of metabolites like 13-Hotre and stearidonic acid. In conclusion, these findings suggest that OP holds promise as a therapeutic agent for the treatment and prevention of S. aureus-induced mastitis.
The tumor microenvironment (TME) is increasingly recognized as a complex ecosystem shaped by dynamic interactions among tumor cells, immune cells, and microbial components. While growing evidence has established the microbiota as a key regulator of antitumor immunity and immunotherapy response, the contribution of bacteriophages, the most abundant biological entities within microbial communities, has remained largely overlooked. Recent studies suggest that bacteriophages are not merely passive regulators of bacterial populations but can actively modulate host immune responses and influence tumor-associated immune landscapes. In this review, we summarize emerging evidence suggesting that bacteriophages may influence antitumor immunity through both direct and indirect mechanisms. Evidence from immune-cell and non-cancer experimental systems indicates that phage nucleic acids can engage TLR9-dependent sensing and, for selected phages, STING-associated inflammatory signaling; however, the relevance of these pathways within human tumors remains to be established. Indirectly, phages may alter microbial community structure and metabolic outputs, which could influence systemic immune tone and the composition of immune infiltrates within the TME. We further discuss accumulating data linking phageome features with tumor progression and responses to immune checkpoint blockade and other cancer therapies. However, much of the available evidence remains preclinical, indirect, or correlative, and causal roles for endogenous phages in human tumor immunity still require further validation. Distinct from the putative ecological and immunological roles of naturally occurring phages, engineered bacteriophages are being developed as therapeutic platforms for cancer immunotherapy, including tumor-antigen display, targeted delivery of immune agonists, cytokines or nucleic acids, and combination strategies with existing treatments. Finally, we address key methodological, mechanistic, and safety challenges that must be overcome to translate phage-based immunomodulation into clinical applications. Collectively, this review highlights the phageome as an emerging regulatory layer of tumor immunity and a promising, yet underexplored, target for therapeutic intervention.
Biochar and metal-resistant bacteria are increasingly used to stabilize toxic metals in contaminated soils. Parthenium biochar and Serratia marcescens were applied in combination to assess their impact on plant growth parameters, physiological and biochemical attributes, metal uptake, soil metal fractions, and oxidative stress responses. Additionally, machine learning models, including support vector machine regression and elastic net, were used to predict and identify key factors influencing metal uptake. The combined treatment significantly enhanced plant biomass, root length, and shoot length by 56-78%. Chlorophyll content increased by 42-68%, membrane injury decreased by 25%, and cell viability improved by 33%, along with increases in proline (45%) and protein contents (35%). Metal uptake was reduced by 40-65%, while bioaccumulation decreased by 40%, and bioconcentration and translocation factors declined by 55% and 60%, respectively. Soil metal fractions (extractable, reducible, and oxidizable) decreased by 30-50%, reducing total metal concentrations by 45%. Integrated Biomarker Response analysis indicated reduced oxidative stress in both leaves and roots, supported by decreased antioxidant enzyme activity. Support vector machine regression showed superior predictive performance (R² = 0.97), whereas elastic net identified key predictors (R² = 0.85). The integrated application of biochar and S. marcescens was associated with improved plant growth, enhanced soil quality, and reduced metal toxicity under multimetal stress. These findings suggest that this approach may have potential for sustainable remediation of contaminated soils.
Base editors (BEs) enable efficient A-to-G or C-to-T conversions without double-stranded DNA cleavage, but their editing windows remain difficult to tune, limiting genome engineering flexibility. Here, we engineered CRISPR/Cas12b sgRNA by introducing MS2 hairpins to recruit an MS2-N55K-cytidine deaminase-UGI complex, enabling programmable control of the editing window. Three modified sgRNAs were generated by replacing two loop regions, each producing distinct editing hotspots in E. coli. The AID*Δ-MSBE system (sgRNA1.1) generated a window near the PAM with peak activity at C7-C9, while the CDA-MSBE system (sgRNA1.2) produced a distal window with peak activity at C20-C23. Both systems exhibited identical editing patterns in Bacillus subtilis. A dual-orthogonal system (MS2 and PP7) was constructed to simultaneously recruit two deaminase complexes, restoring the classic dCas12b CBE editing pattern. Rifampicin resistance assays confirmed high targeting specificity with low off-target effects. As proof of concept, the MSBEs were successfully employed for the flexible reprogramming of sfGFP fluorescence and the targeted evolution of the endogenous gene rpsE, respectively. Collectively, we developed the MSBEs with tunable editing hotspots, providing innovative tools to enhance the flexibility and accessibility of BEs for genome engineering.
White-nose syndrome, caused by Pseudogymnoascus destructans (Pd), is a major fungal disease threatening hibernating bats. Cave soils can serve as environmental reservoirs for Pd, yet the microbial and biochemical mechanisms underlying naturally low Pd burdens in some cave environments remain poorly understood. Here, we integrated soil microbiome profiling, metagenomics, metabolomics, multi-omics network analysis, and in vitro validation to investigate the ecological and functional basis of differential Pd loads in hibernating bat caves in Northeast China. The three caves shared cold, humid, and weakly acidic microenvironments, but differed significantly in electrical conductivity, soil water content, nutrient availability, and extracellular enzyme activities. Soil microbial communities showed significant inter-cave variation in composition, diversity, and niche breadth, with stochastic processes contributing substantially to community assembly. Environmental variables, particularly pH and Pd load, were important predictors of microbial community structure. Functional analyses revealed that the low-Pd Gezi Cave was enriched in genes associated with organic carbon degradation, nitrogen input and retention, and secondary metabolism. Metabolomic profiling further identified cave-specific metabolite signatures, among which Biochanin A, 4-Hydroxybenzaldehyde, Vanillin, and Arachidonic acid were negatively correlated with Pd loads. Integrated pathway and network analyses showed that differential genes and metabolites jointly mapped to secondary metabolite biosynthesis, aminobenzoate degradation, and flavonoid degradation pathways, forming a microbe-metabolite-functional gene coupling network involving key taxa such as Rhodococcus, Pseudorhodoplanes, and Rhodoplanes. In vitro assays confirmed that 4-Hydroxybenzaldehyde, Coumarin, and Vanillin inhibited Pd growth. Structural equation modelling further indicated that environmental heterogeneity was associated with variation in Pd loads through microbial functional attributes and metabolite profiles. These findings suggest that naturally low-Pd cave soils are associated with coordinated environmental filtering, microbial functional specialization, and antifungal metabolite production, providing mechanistic insight into microbial and biochemical constraints on Pd persistence in cave reservoirs.
