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Abstract Few aerobic hyperthermophilic microorganisms degrade polysaccharides. Here, we describe the genome-enabled enrichment and optical tweezer-based isolation of an aerobic polysaccharide-degrading hyperthermophile, Fervidibacter sacchari, previously ascribed to candidate phylum Fervidibacteria. F. sacchari uses polysaccharides and monosaccharides for growth at 65–87.5 °C and expresses 191 carbohydrate-active enzymes (CAZymes) according to RNA-Seq and proteomics, including 31 with unusual glycoside hydrolase domains (GH109, GH177, GH179). Fluorescence in-situ hybridization and nanoscale secondary ion mass spectrometry confirmed rapid assimilation of 13C-starch in spring sediments. Purified GHs were optimally active at 80–100 °C on ten different polysaccharides. Finally, we propose reassigning Fervidibacteria as a class within phylum Armatimonadota, along with 18 other species, and show that a high number and diversity of CAZymes is a hallmark of the phylum, in both aerobic and anaerobic lineages. Our study establishes Fervidibacteria as hyperthermophilic polysaccharide degraders in terrestrial geothermal springs and suggests a broad role for Armatimonadota in polysaccharide catabolism.

Abstract Background The Atribacterota are widely distributed in the subsurface biosphere. Recently, the first Atribacterota isolate was described and the number of Atribacterota genome sequences retrieved from environmental samples has increased significantly; however, their diversity, physiology, ecology, and evolution remain poorly understood. Results We report the isolation of the second member of Atribacterota, Thermatribacter velox gen. nov., sp. nov., within a new family Thermatribacteraceae fam. nov., and the short-term laboratory cultivation of a member of the JS1 lineage, Phoenicimicrobium oleiphilum HX-OS.bin.34TS, both from a terrestrial oil reservoir. Physiological and metatranscriptomics analyses showed that Thermatribacter velox B11T and Phoenicimicrobium oleiphilum HX-OS.bin.34TS ferment sugars and n-alkanes, respectively, producing H2, CO2, and acetate as common products. Comparative genomics showed that all members of the Atribacterota lack a complete Wood-Ljungdahl Pathway (WLP), but that the Reductive Glycine Pathway (RGP) is widespread, indicating that the RGP, rather than WLP, is a central hub in Atribacterota metabolism. Ancestral character state reconstructions and phylogenetic analyses showed that key genes encoding the RGP (fdhA, fhs, folD, glyA, gcvT, gcvPAB, pdhD) and other central functions were gained independently in the two classes, Atribacteria (OP9) and Phoenicimicrobiia (JS1), after which they were inherited vertically; these genes included fumarate-adding enzymes (faeA; Phoenicimicrobiia only), the CODH/ACS complex (acsABCDE), and diverse hydrogenases (NiFe group 3b, 4b and FeFe group A3, C). Finally, we present genome-resolved community metabolic models showing the central roles of Atribacteria (OP9) and Phoenicimicrobiia (JS1) in acetate- and hydrocarbon-rich environments. Conclusion Our findings expand the knowledge of the diversity, physiology, ecology, and evolution of the phylum Atribacterota. This study is a starting point for promoting more incisive studies of their syntrophic biology and may guide the rational design of strategies to cultivate them in the laboratory. Video Abstract

Abstract Terrestrial geothermal springs are physicochemically diverse and host abundant populations of Archaea. However, the diversity, functionality, and geological influences of these Archaea are not well understood. Here we explore the genomic diversity of Archaea in 152 metagenomes from 48 geothermal springs in Tengchong, China, collected from 2016 to 2021. Our dataset is comprised of 2949 archaeal metagenome-assembled genomes spanning 12 phyla and 392 newly identified species, which increases the known species diversity of Archaea by ~48.6%. The structures and potential functions of the archaeal communities are strongly influenced by temperature and pH, with high-temperature acidic and alkaline springs favoring archaeal abundance over Bacteria. Genome-resolved metagenomics and metatranscriptomics provide insights into the potential ecological niches of these Archaea and their potential roles in carbon, sulfur, nitrogen, and hydrogen metabolism. Furthermore, our findings illustrate the interplay of competition and cooperation among Archaea in biogeochemical cycles, possibly arising from overlapping functional niches and metabolic handoffs. Taken together, our study expands the genomic diversity of Archaea inhabiting geothermal springs and provides a foundation for more incisive study of biogeochemical processes mediated by Archaea in geothermal ecosystems.

In recent years, there has been an increasing focus on microbial ecology and its possible impact on agricultural production, owing to its eco-friendly nature and sustainable use. The current study employs metabolomics technologies and bioinformatics approaches to identify changes in the exometabolome of Streptomyces albidoflavus B24. This research aims to shed light on the mechanisms and metabolites responsible for the antifungal and growth promotion strategies, with potential applications in sustainable agriculture. Metabolomic analysis was conducted using Q Exactive UPLC–MS/MS. Our findings indicate that a total of 3,840 metabolites were identified, with 137 metabolites exhibiting significant differences divided into 61 up and 75 downregulated metabolites based on VIP >1, |FC| >1, and p

Abstract Background The microbiome of the Sinai Desert farming system plays an important role in the adaptive strategy of growing crops in a harsh, poly-extreme, desert environment. However, the diversity and function of microbial communities under this unfavorable moisture and nutritional conditions have not yet been investigated. Based on culturomic and metagenomic methods, we analyzed the microbial diversity and function of a total of fourteen rhizosphere soil samples (collected from twelve plants in four farms of the Sinai desert), which may provide a valuable and meaningful guidance for the design of microbial inoculants. Results The results revealed a wide range of microbial taxa, including a high proportion of novel undescribed lineages. The composition of the rhizosphere microbial communities differed according to the sampling sites, despite similarities or differences in floristics. Whereas, the functional features of rhizosphere microbiomes were significantly similar in different sampling sites, although the microbial communities and the plant hosts themselves were different. Importantly, microorganisms involved in ecosystem functions are different between the sampling sites, for example nitrogen fixation was prevalent in all sample sites while microorganisms responsible for this process were different. Conclusion Here, we provide the first characterization of microbial communities and functions of rhizosphere soil from the Sinai desert farming systems and highlight its unexpectedly high diversity. This study provides evidence that the key microorganisms involved in ecosystem functions are different between sampling sites with different environment conditions, emphasizing the importance of the functional microbiomes of rhizosphere microbial communities. Furthermore, we suggest that microbial inoculants to be used in future agricultural production should select microorganisms that can be involved in plant-microorganism interactions and are already adapted to a similar environmental setting.

Abstract Background Candidatus Nanohaloarchaeota, an archaeal phylum within the DPANN superphylum, is characterized by limited metabolic capabilities and limited phylogenetic diversity and until recently has been considered to exclusively inhabit hypersaline environments due to an obligate association with Halobacteria. Aside from hypersaline environments, Ca. Nanohaloarchaeota can also have been discovered from deep-subsurface marine sediments. Results Three metagenome-assembled genomes (MAGs) representing a new order within the Ca. Nanohaloarchaeota were reconstructed from a stratified salt crust and proposed to represent a novel order, Nucleotidisoterales. Genomic features reveal them to be anaerobes capable of catabolizing nucleotides by coupling nucleotide salvage pathways with lower glycolysis to yield free energy. Comparative genomics demonstrated that these and other Ca. Nanohaloarchaeota inhabiting saline habitats use a “salt-in” strategy to maintain osmotic pressure based on the high proportion of acidic amino acids. In contrast, previously described Ca. Nanohaloarchaeota MAGs from geothermal environments were enriched with basic amino acids to counter heat stress. Evolutionary history reconstruction revealed that functional differentiation of energy conservation strategies drove diversification within Ca. Nanohaloarchaeota, further leading to shifts in the catabolic strategy from nucleotide degradation within deeper lineages to polysaccharide degradation within shallow lineages. Conclusions This study provides deeper insight into the ecological functions and evolution of the expanded phylum Ca. Nanohaloarchaeota and further advances our understanding on the functional and genetic associations between potential symbionts and hosts. Video Abstract

Abstract The great majority of microorganisms are as‐yet‐uncultivated, mostly found in extreme environments. High‐throughput sequencing provides data‐rich genomes from single‐cell and metagenomic techniques, which has enabled researchers to obtain a glimpse of the unexpected genetic diversity of “microbial dark matter.” However, cultivating microorganisms from extreme environments remains essential for dissecting and utilizing the functions of extremophiles. Here, we provide a straightforward protocol for efficiently isolating prokaryotic microorganisms from different extreme habitats (thermal, xeric, saline, alkaline, acidic, and cryogenic environments), which was established through previous successful work and our long‐term experience in extremophile resource mining. We propose common processes for extremophile isolation at first and then summarize multiple cultivation strategies for recovering prokaryotic microorganisms from extreme environments and meanwhile provide specific isolation tips that are always overlooked but important. Furthermore, we propose the use of multi‐omics‐guided microbial cultivation approaches for culturing these as‐yet‐uncultivated microorganisms and two examples are provided to introduce how these approaches work. In summary, the protocol allows researchers to significantly improve the isolation efficiency of pure cultures and novel taxa, which therefore paves the way for the protection and utilization of microbial resources from extreme environments.

Trace metals have been an important ingredient for life throughout Earth’s history. Here, the authors show that a member of an elusive archaeal lineage (Caldarchaeales or Aigarchaeota) requires tungsten for growth, and provide evidence that tungsten-dependent metabolism played a role in the origin and evolution of this lineage.

Abstract Biological denitrification is a crucial process in the nitrogen biogeochemical cycle, and Thermus has been reported to be a significant heterotrophic denitrifier in terrestrial geothermal environments. However, neither the denitrification potential nor the evolutionary history of denitrification genes in the genus Thermus or phylum Deinococcota is well understood. Here, we performed a comparative analysis of 23 Thermus genomes and identified denitrification genes in 15 Thermus strains. We confirmed that Thermus harbors an incomplete denitrification pathway as none of the strains contain the nosZ gene. Ancestral character state reconstructions and phylogenetic analyses showed that narG, nirS, and norB genes were acquired by the last common ancestor of Thermales and were inherited vertically. In contrast, nirK of Thermales was acquired via two distinct horizontal gene transfers from Proteobacteria to the genus Caldithermus and from an unknown donor to the common ancestor of all known Thermus species except Thermus filiformis. This study expands our understanding of the genomic potential for incomplete denitrification in Thermus, revealing a largely vertical evolutionary history of the denitrification pathway in the Thermaceae, and supporting the important role for Thermus as an important heterotrophic denitrifier in geothermal environments.

ABSTRACT “Candidatus Parvarchaeales” microbes, representing a DPANN archaeal group with limited metabolic potential and reliance on hosts for their growth, were initially found in acid mine drainage (AMD). Due to the lack of representatives, however, their ecological roles and adaptation to extreme habitats such as AMD as well as how they diverge across the lineage remain largely unexplored. By applying genome-resolved metagenomics, 28 Parvarchaeales-associated metagenome-assembled genomes (MAGs) representing two orders and five genera were recovered. Among them, we identified three new genera and proposed the names “Candidatus Jingweiarchaeum,” “Candidatus Haiyanarchaeum,” and “Candidatus Rehaiarchaeum,” with the former two belonging to a new order, “Candidatus Jingweiarchaeales.” Further analyses of the metabolic potentials revealed substantial niche differentiation between Jingweiarchaeales and Parvarchaeales. Jingweiarchaeales may rely on fermentation, salvage pathways, partial glycolysis, and the pentose phosphate pathway (PPP) for energy conservation reservation, while the metabolic potentials of Parvarchaeales might be more versatile. Comparative genomic analyses suggested that Jingweiarchaeales favor habitats with higher temperatures and that Parvarchaeales are better adapted to acidic environments. We further revealed that the thermal adaptation of these lineages, especially Haiyanarchaeum, might rely on genomic features such as the usage of specific amino acids, genome streamlining, and hyperthermophile featured genes such as rgy. Notably, the adaptation of Parvarchaeales to acidic environments was possibly driven by horizontal gene transfer (HGT). The reconstruction of ancestral states demonstrated that both may have originated from thermal and neutral environments and later spread to mesothermal and acidic environments. These evolutionary processes may also be accompanied by adaptation to oxygen-rich environments via HGT. IMPORTANCE “Candidatus Parvarchaeales” microbes may represent a lineage uniquely distributed in extreme environments such as AMD and hot springs. However, little is known about the strategies and processes of how they adapted to these extreme environments. By the discovery of potential new order-level lineages, “Ca. Jingweiarchaeales,” and in-depth comparative genomic analysis, we unveiled the functional differentiation of these lineages. Furthermore, we show that the adaptation of these lineages to high-temperature and acidic environments was driven by different strategies, with the former relying more on genomic characteristics such as genome streamlining and amino acid compositions and the latter relying more on the acquisition of genes associated with acid tolerance. Finally, by the reconstruction of the ancestral states of the optimal growth temperature (OGT) and isoelectric point (pI), we showed the potential evolutionary process of Parvarchaeales-related lineages with regard to the shift from the high-temperature environment of their common ancestors to low-temperature (potentially acidic) environments.

Anaerobic ammonium oxidation (anammox) is an important process of the nitrogen cycle, and the anammox bacteria have been studied in a wide variety of environments. However, the distribution, diversity, and abundance of anammox bacteria in hot springs remain enigmatic. In this study, the anammox process was firstly investigated in hot springs of Conghua, China. Anammox-like bacterial sequences that closely affiliated to “Candidatus Brocadia,” “Candidatus Kuenenia,” “Candidatus Scalindua,” “Candidatus Anammoxoglobus,” and “Candidatus Jettenia” were detected. Several operational taxonomic units (OTUs) from this study shared low sequence identities to the 16S rRNA gene of the known anammox bacteria, suggesting that they might be representing putative novel anammox bacteria. A quantitative PCR analysis of anammox-specific 16S rRNA gene confirmed that the abundance of anammox bacteria ranged from 1.60 × 104 to 1.20 × 107 copies L–1. Nitrate was a key environmental factor defining the geographical distribution of the anammox bacterial community in the hot spring ecosystem. Dissolved inorganic carbon had a significant influence on anammox bacterial biodiversity. Our findings for the first time revealed that the diverse anammox bacteria, including putative novel anammox bacterial candidates, were present in Conghua hot spring, which extended the existence of anammox bacteria to the hot springs in China and expands our knowledge of the biogeography of anammox bacteria. This work filled up the research lacuna of anammox bacteria in Chinese hot spring habitat and would guide for enrichment strategies of anammox bacteria of Conghua hot springs.

The authors build a reference phylogeny of 10,575 evenly-sampled bacterial and archaeal genomes, based on 381 markers. The results indicate a remarkably closer evolutionary proximity between Archaea and Bacteria than previous estimates that used fewer “core” genes, such as the ribosomal proteins.

ABSTRACT “Candidatus Bathyarchaeia” is a phylogenetically diverse and widely distributed lineage often in high abundance in anoxic submarine sediments; however, their evolution and ecological roles in terrestrial geothermal habitats are poorly understood. In the present study, 35 Ca. Bathyarchaeia metagenome-assembled genomes (MAGs) were recovered from hot spring sediments in Tibet and Yunnan, China. Phylogenetic analysis revealed all MAGs of Ca. Bathyarchaeia can be classified into 7 orders and 15 families. Among them, 4 families have been first discovered in the present study, significantly expanding the known diversity of Ca. Bathyarchaeia. Comparative genomics demonstrated Ca. Bathyarchaeia MAGs from thermal habitats to encode a large variety of genes related to carbohydrate degradation, which are likely a metabolic adaptation of these organisms to a lifestyle at high temperatures. At least two families are potential methanogens/alkanotrophs, indicating a potential for the catalysis of short-chain hydrocarbons. Three MAGs from Family-7.3 are identified as alkanotrophs due to the detection of an Mcr complex. Family-2 contains the largest number of genes relevant to alkyl-CoM transformation, indicating the potential for methylotrophic methanogenesis, although their evolutionary history suggests the ancestor of Ca. Bathyarchaeia was unable to metabolize alkanes. Subsequent lineages have acquired the ability via horizontal gene transfer. Overall, our study significantly expands our knowledge and understanding of the metabolic capabilities, habitat adaptations, and evolution of Ca. Bathyarchaeia in thermal environments. IMPORTANCE Ca. Bathyarchaeia MAGs from terrestrial hot spring habitats are poorly revealed, though they have been studied extensively in marine ecosystems. In this study, we uncovered the metabolic capabilities and ecological role of Ca. Bathyarchaeia in hot springs and give a comprehensive comparative analysis between thermal and nonthermal habitats to reveal the thermal adaptability of Ca. Bathyarchaeia. Also, we attempt to determine the evolutionary history of methane/alkane metabolism in Ca. Bathyarchaeia, since it appears to be the first archaea beyond Euryarchaeota which contains the mcrABG genes. The reclassification of Ca. Bathyarchaeia and significant genomic differences among different lineages largely expand our knowledge on these cosmopolitan archaea, which will be beneficial in guiding the future studies.

ABSTRACT “Candidatus Aenigmarchaeota” (“Ca. Aenigmarchaeota”) represents one of the earliest proposed evolutionary branches within the Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, and Nanohaloarchaeota (DPANN) superphylum. However, their ecological roles and potential host-symbiont interactions are still poorly understood. Here, eight metagenome-assembled genomes (MAGs) were reconstructed from hot spring ecosystems, and further in-depth comparative and evolutionary genomic analyses were conducted on these MAGs and other genomes downloaded from public databases. Although with limited metabolic capacities, we reported that “Ca. Aenigmarchaeota” in thermal environments harbor more genes related to carbohydrate metabolism than “Ca. Aenigmarchaeota” in nonthermal environments. Evolutionary analyses suggested that members from the Thaumarchaeota, Aigarchaeota, Crenarchaeota, and Korarchaeota (TACK) superphylum and Euryarchaeota contribute substantially to the niche expansion of “Ca. Aenigmarchaeota” via horizontal gene transfer (HGT), especially genes related to virus defense and stress responses. Based on co-occurrence network results and recent genetic exchanges among community members, we conjectured that “Ca. Aenigmarchaeota” may be symbionts associated with one MAG affiliated with the genus Pyrobaculum, though host specificity might be wide and variable across different “Ca. Aenigmarchaeota” organisms. This study provides significant insight into possible DPANN-host interactions and ecological roles of “Ca. Aenigmarchaeota.” IMPORTANCE Recent advances in sequencing technology promoted the blowout discovery of super tiny microbes in the Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, and Nanohaloarchaeota (DPANN) superphylum. However, the unculturable properties of the majority of microbes impeded our investigation of their behavior and symbiotic lifestyle in the corresponding community. By integrating horizontal gene transfer (HGT) detection and co-occurrence network analysis on “Candidatus Aenigmarchaeota” (“Ca. Aenigmarchaeota”), we made one of the first attempts to infer their putative interaction partners and further decipher the potential functional and genetic interactions between the symbionts. We revealed that HGTs contributed by members from the Thaumarchaeota, Aigarchaeota, Crenarchaeota, and Korarchaeota (TACK) superphylum and Euryarchaeota conferred “Ca. Aenigmarchaeota” with the ability to survive under different environmental stresses, such as virus infection, high temperature, and oxidative stress. This study demonstrates that the interaction partners might be inferable by applying informatics analyses on metagenomic sequencing data.

Methane metabolism by some lineages of Archaea contributes to the cycling of carbon on Earth. Here, the authors show high diversity of methyl-coenzyme M reductase (Mcr), a key enzyme associated with archaeal methane/alkane metabolism, in hot spring Archaea, and investigate their ecological roles and evolution.

Thermoflexus hugenholtzii JAD2T, the only cultured representative of the Chloroflexota order Thermoflexales, is abundant in Great Boiling Spring (GBS), NV, United States, and close relatives inhabit geothermal systems globally. However, no defined medium exists for T. hugenholtzii JAD2T and no single carbon source is known to support its growth, leaving key knowledge gaps in its metabolism and nutritional needs. Here, we report comparative genomic analysis of the draft genome of T. hugenholtzii JAD2T and eight closely related metagenome-assembled genomes (MAGs) from geothermal sites in China, Japan, and the United States, representing “Candidatus Thermoflexus japonica,” “Candidatus Thermoflexus tengchongensis,” and “Candidatus Thermoflexus sinensis.” Genomics was integrated with targeted exometabolomics and 13C metabolic probing of T. hugenholtzii. The Thermoflexus genomes each code for complete central carbon metabolic pathways and an unusually high abundance and diversity of peptidases, particularly Metallo- and Serine peptidase families, along with ABC transporters for peptides and some amino acids. The T. hugenholtzii JAD2T exometabolome provided evidence of extracellular proteolytic activity based on the accumulation of free amino acids. However, several neutral and polar amino acids appear not to be utilized, based on their accumulation in the medium and the lack of annotated transporters. Adenine and adenosine were scavenged, and thymine and nicotinic acid were released, suggesting interdependency with other organisms in situ. Metabolic probing of T. hugenholtzii JAD2T using 13C-labeled compounds provided evidence of oxidation of glucose, pyruvate, cysteine, and citrate, and functioning glycolytic, tricarboxylic acid (TCA), and oxidative pentose-phosphate pathways (PPPs). However, differential use of position-specific 13C-labeled compounds showed that glycolysis and the TCA cycle were uncoupled. Thus, despite the high abundance of Thermoflexus in sediments of some geothermal systems, they appear to be highly focused on chemoorganotrophy, particularly protein degradation, and may interact extensively with other microorganisms in situ.

The bioconversion of lignocellulose in various industrial processes, such as biofuel production, requires the degradation of cellulose. Actinomadura amylolytica YIM 77502T is an aerobic, Gram-positive actinomycete that can efficiently degrade crystalline cellulose by extracellular cellulases. Genomic analysis of A. amylolytica identified 9 cellulase and 11 β-glucosidase genes that could potentially encode proteins that digest cellulose. Extracellular proteome characterization of A. amylolytica cell-free culture supernatant by liquid chromatography tandem mass spectrometry analysis revealed that 4 of these cellulases and 2 of these β-glucosidases functioned during cellulose hydrolysis. Thin-layer chromatography analysis revealed extracellular β-glucosidases play a major role in carboxyl methyl cellulose (CMC) degradation of products in culture supernatants. In this study, 2 of the identified secreted β-glucosidases, AaBGL1 and AaBGL2, were functionally expressed in Escherichia coli and found to have β-glucosidase activity with wide substrate specificities, including for p-nitrophenyl β-D-glucopyranoside (pNPG), p-nitrophenyl-beta-D-cellobioside (pNPC), and cellobiose. Moreover, AaBGL1 and AaBGL2 had high tolerances for glucose. After adding these β-glucosidases to commercial cellulases, the degradation rates of CMC, Avicel, birch sawdust, and corncob powder increased by 37, 42, 33, and 9%, respectively. Overall, this work identifies an alternative potential source of β-glucosidases with potential applications in commercial cellulose utilization and the bioenergy industry.

The phylum Actinobacteria is one of the most ubiquitously present bacterial lineages on Earth. In the present study, we try to explore the diversity of cultivable rare Actinobacteria in Sigangli Cave, Yunnan, China by utilizing a combination of different sample pretreatments and under different culture conditions. Pretreating the samples under different conditions of heat, setting the isolation condition at different pHs, and supplementation of media with different calcium salts were found to be effective for isolation of diverse rare Actinobacteria. During our study, a total of 204 isolates affiliated to 30 genera of phylum Actinobacteria were cultured. Besides the dominant Streptomyces, rare Actinobacteria of the genera Actinocorallia, Actinomadura, Agromyces, Alloactinosynnema, Amycolatopsis, Beutenbergia, Cellulosimicrobium, Gordonia, Isoptericola, Jiangella, Knoellia, Kocuria, Krasilnikoviella, Kribbella, Microbacterium, Micromonospora, Mumia, Mycobacterium, Nocardia, Nocardioides, Nocardiopsis, Nonomuraea, Oerskovia, Pseudokineococcus, Pseudonocardia, Rhodococcus, Saccharothrix, Streptosporangium, and Tsukamurella were isolated from these cave samples.

In recent years, there has been an increasing focus on microbial ecology and its possible impact on agricultural production, owing to its eco-friendly nature and sustainable use. The current study employs metabolomics technologies and bioinformatics approaches to identify changes in the exometabolome of Streptomyces albidoflavus B24. This research aims to shed light on the mechanisms and metabolites responsible for the antifungal and growth promotion strategies, with potential applications in sustainable agriculture. Metabolomic analysis was conducted using Q Exactive UPLC-MS/MS. Our findings indicate that a total of 3,840 metabolites were identified, with 137 metabolites exhibiting significant differences divided into 61 up and 75 downregulated metabolites based on VIP >1, |FC| >1, and p < 0.01. The interaction of S. albidoflavus B24 monoculture with the co-culture demonstrated a stronger correlation coefficient. The Principal Component Analysis (PCA) demonstrates that PCA1 accounted for 23.36%, while PCA2 accounted for 20.28% distinction. OPLS-DA score plots indicate significant separation among different groups representing (t1) 24% as the predicted component (to1) depicts 14% as the orthogonal component. According to the findings of this comprehensive study, crude extracts from S. albidoflavus demonstrated varying abilities to impede phytopathogen growth and enhance root and shoot length in tested plants. Through untargeted metabolomics, we discovered numerous potential molecules with antagonistic activity against fungal phytopathogens among the top 10 significant metabolites with the highest absolute log2FC values. These include Tetrangulol, 4-Hydroxybenzaldehyde, and Cyclohexane. Additionally, we identified plant growth-regulating metabolites such as N-Succinyl-L-glutamate, Nicotinic acid, L-Aspartate, and Indole-3-acetamide. The KEGG pathway analysis has highlighted these compounds as potential sources of antimicrobial properties The inhibitory effect of S. albidoflavus crude extracts on pathogen growth is primarily attributed to the presence of specific gene clusters responsible for producing cyclic peptides such as ansamycins, porphyrin, alkaloid derivatives, and neomycin. Overall, it is apparent that crude extracts from S. albidoflavus exhibited varying abilities to inhibit the growth of three phytopathogens and enhancement in both root and shoot length of tested plants. This research enhances our understanding of how secondary metabolites contribute to growth promotion and biocontrol, supporting ecosystem sustainability and resilience while boosting productivity in sustainable agriculture. [ABSTRACT FROM AUTHOR]