Abstract:Chitin is the second most abundant natural biopolymer on Earth after cellulose, primarily derived from the exoskeletons of crustaceans. With the rapid development of the seafood processing industry, large amounts of chitin-rich waste are being generated. Chitinases, which catalyze the hydrolysis of chitin, are widely distributed across all three domains of life and can degrade chitin into chitooligosaccharides or monosaccharides with diverse biological activities. Archaea, as prokaryotes that typically inhabit extreme environments, produce chitinases with exceptional stability and tolerance, demonstrating unique advantages in industrial catalysis and biotransformation. However, studies on archaeal chitinases remain limited. This review systematically elucidates the enzymatic degradation process of chitin and the catalytic mechanisms of chitinases, with a particular focus on the domain organization and enzymatic properties of archaeal chitinases. In addition, we analyze the distribution and classification of archaeal chitinases and discuss their potential applications in biomanufacturing, medicine, and agricultural biocontrol. Moreover, we summarize the current limitations in functional studies of archaeal chitinases and point out the key challenges and future directions for promoting industrial applications.

Abstract:Coss-assembly-like phages (crAss-like phages), a group of double-stranded DNA viruses identified through cross-assembly analysis of human fecal metagenomic data, are classified within the order Crassvirales and predominantly exhibit tailed morphology. As ubiquitous and stable components of the human gut, crAss-like phages represent core members of the gut virome. These phages exhibit diverse infection strategies toward hosts (predominantly Bacteroidetes), ranging from non-lytic persistent infection and multiple rounds of lytic infection to delayed lysis. Functioning as key nodes linking gut microbial ecology with host metabolic homeostasis, crAss-like phages have thus emerged as critical regulators in gut ecosystem dynamics. This review systematically summarizes current knowledge regarding the discovery and taxonomy, genomic structure characteristics, available isolates, host-phage interactions, and health implications of crAss-like phages. Furthermore, we outline the future research directions that position these phages as a foundational “viral cornerstone” in gut ecosystem studies, aiming to provide a conceptual framework and novel insights for subsequent investigations in this field.

Abstract:As a globally important economic crop, the quality and growth of tobacco (Nicotiana tabacum L.) depends on the regulatory role of the symbiotic microbial community. Taking “core microbiome” as a starting point, we elaborate on the key roles of tobacco-related microorganisms: (1) as an important component of the tobacco holobiont, the composition of the tobacco core microbiome and its dynamic changes in different parts and developmental stages of tobacco; (2) the microbial community promoting nutrient acquisition, enhancing stress resistance, and regulating growth to maintain the health of tobacco crops; (3) the contribution of microbial activities, such as carbohydrate degradation and alkaloid conversion, to the formation of tobacco flavor during post-harvest processes like curing, fermentation, and aging. Prospects require integrating multi-omics and field experiments to elucidate the functional mechanisms of tobacco core microbiome, aiming to develop targeted regulatory strategies. This review provides a theoretical basis for optimizing tobacco cultivation and quality through microbiome regulation, providing scientific guidance for tobacco production.

Abstract:Microorganisms play crucial roles in food, agriculture, medicine, and environmental sectors due to their remarkable ecological adaptability and functional diversity. However, only a limited number of microbial species can be cultivated under conventional laboratory conditions, leaving vast microbial diversity yet to be studied and utilized. Recent innovations, such as in situ cultivation using diffusion chambers and high-throughput sorting/cultivation via droplet microfluidics, have significantly advanced the isolation and cultivation of uncultivable microorganisms. Nevertheless, existing technologies primarily focus on initial isolation and primary cultivation, with insufficient attention to subculturing, the critical phase determining pure culture success. This gap is particularly pronounced for marine microorganisms, whose unique habitats and metabolic traits impose stringent demands on culture media. The progress in droplet microfluidics has increased the isolation flux and saved labor, while the success of subculturing relies more on the innovation of culture media. Synthesizing prior research with experience on marine organism-associated bacteria, this work proposes “droplet microfluidics+culture medium design and optimization” as the breakthrough strategy for future cultivation of marine uncultivable microorganisms.

Abstract:As an important part of the grassland ecosystem, desert steppes account for about 8.1% of the total grassland area. They represent the most special and challenging ecological environments due to the unique plant community composition, structural types, and ecological functions. Desert steppes not only have a variety of natural geographical backgrounds and ecological functions, but also play an important role in global climate change responses and biodiversity conservation. As a key biological component driving soil nutrient cycling, organic matter decomposition, and soil structure formation, soil microorganisms play an essential role in maintaining ecosystem functions. The ways of soil microorganisms adapting to harsh habitats and maintaining their ecological functions have become a scientific issue worthy of further study, especially in desert steppes. This paper systematically introduces the characteristics of soil microbial community composition in desert steppes and comprehensively reviews its response mechanisms to key factors of global climate change (temperature and precipitation changes, nitrogen deposition, and elevated carbon dioxide concentration) and abiotic stress factors (drought, saline-alkali stress, and heavy metal stress). Through this review, we aim to understand the ecological functions and adaptation strategies of soil microorganisms in desert steppes and provide a scientific basis for the protection and sustainable management of such fragile ecosystems.

Abstract:Background Alginate lyase is a specialized polysaccharide lyase that is crucial for the degradation of algin into alginic oligosaccharides (AOS), demonstrating promising applications across agriculture, food production, and pharmaceuticals. However, the natural form of alginate lyase suffers from inherent limitations, including poor thermal stability and low enzymatic activity. These shortcomings pose barriers to its large-scale industrial production and broader application.Objective To develop an alginate lyase mutant with significantly enhanced activity and thermal stability, thus facilitating its industrial production and advancing the use of enzymatic preparations of seaweed fertilizers.Methods To enhance the thermal stability of the alginate lyase derived from Marinimicrobium sp. H1, we employed a rational design strategy in conjunction with ΔΔG folding evaluation. The efficient expression of the mutant, designated AlgH1-T140Q, was achieved in a Bacillus subtilis expression system, through the optimization of components such as the promoter and signal peptide as well as fermentation conditions.Results AlgH1-T140Q exhibited a remarkable improvement of 25.70% in thermal stability compared with the wild-type enzyme. Screening efforts led to the identification of the optimal expression promoter, P₄₃, and the optimal signal peptide, SP_lipA, resulting in a substantial increase of 235% in extracellular enzyme activity. Furthermore, through meticulous optimization of the fermentation process, the AlgH1-T140Q activity in a 20-L pilot-scale fermentation system was 304% greater than that in initial shake flask experiments. Moreover, the engineered enzyme reached the degradation rates of 89.23%, 74.05%, and 76.80% for Laminaria, Ascophyllum, and Sargassum, respectively.Conclusion The findings of this study demonstrate significant enhancements in both thermal stability and enzymatic activity of AlgH1-T140Q. These advancements indicate strong potential for the application of enzymatic methods in the production of algae-based fertilizers.

Abstract:Background In the acid mine drainage (AMD) treatment system using alkaline slurry, sulfate reducing bacteria (SRB) contained in alkaline slurry could be introduced into the system and participate in the transformation of SO₄^2- and metallic substances. This process is susceptible to environmental conditions including pH value, organic substances, and suspended solid (SS) concentration.Objective The bacterial responses to environmental conditions during this process require further clarification.Methods In the current study, the characteristics of bacterial community structures in the wastewater treatment system, AMD, and slurry were analyzed. On the basis of microbial analysis, the influences of environmental factors and microbial interactions on the growth and metabolism of SRB in the AMD treatment system were elucidated.Results The results demonstrated that the microbial diversity in the treatment system was superior to that in AMD and alkaline slurry. The dominant SRB were affiliated to Desulfovibrio and Desulfitobacterium, the total relative abundance of which reached 3.3%-3.8%. The microorganisms with the functions of decomposing refractory organic matter and promoting Zoogloea formation mitigated the inhibitory effects of external toxic substances on SRB. Moreover, the mixed microbial flora promoted the microbial conversion activity of carbon, nitrogen, and sulfur substances by enhancing metabolic functions such as substance transport and quorum sensing, thereby elevating the transformation activity of SO₄^2- and metallic substances.Conclusion The microbial mass exchange and existing of SS enhanced the stable growth and metabolism of SRB. This study provides a theoretical basis for optimizing AMD treatment systems using biological approaches and supporting the protection of mining environments.

Abstract:Background Photobacterium damselae is a resident bacterium in the shrimp gut and can cause various shrimp diseases. However, systematic research on the biological characteristics and potential pathogenicity of gut-derived P. damselae in shrimps remains lacking.Objective To provide a basis for prevention and utilization of this bacterium in shrimp aquaculture, we isolated, identified, and characterized P. damselae from the gut of the Pacific white shrimp (Litopenaeus vannamei).Methods Photobacterium strains were isolated and screened from the gut of L. vannamei sampled across six farming areas in Zhanjiang, Guangdong. Representative strains were identified based on morphological, physiological, and biochemical characteristics and ureC sequence. Their hemolytic activity and extracellular enzyme production were tested via the plate method. The resistance of the strains to 18 antimicrobial agents was evaluated via the disk diffusion method, and their virulence and potential pathogenicity were assessed by detecting typical virulence genes and conducting toxicity assays in Artemia franciscana larvae.Results P. damselae subsp. damselae (Pdd) was isolated from the guts of shrimps across different farming areas. Except differences in the utilization of sucrose, mannitol, cellobiose, and galactose, the physiological and biochemical characteristics of the 16 representative strains were consistent. Among the 16 strains, 7 showed α-hemolysis, while 9 exhibited β-hemolysis. All the strains produced extracellular lipase and lecithinase, with 11 of them also secreting chitinase. However, differences were observed in their enzymatic activities. The multidrug resistance rate of the 16 strains was as high as 87.5%. Moreover, at least 50.0% of the strains were resistant to ampicillin, amoxicillin, streptomycin, and furazolidone. All Pdd strains carried the phospholipase gene plpV, and the β-hemolytic strains all carried the hemolysin gene hlyAch. Additionally, four strains possessed the collagenase gene colP, while the virulence plasmid pPHDD1 and its related hemolysin genes dly and hlyApl were not detected in any of the strains. Except two Pdd strains that showed no obvious toxicity to A. franciscana larvae, the remaining 14 strains exhibited lethal effects (P<0.05).Conclusion Pdd is commonly found in the gut of L. vannamei, with the vast majority of strains exhibiting visible extracellular enzyme activity and multidrug resistance, as well as significant lethality to A. franciscana larvae, posing a potentially high pathogenic risk to L. vannamei.

Abstract:Background Bioleaching of sulfide ores by acidophilic microorganisms is an effective approach for metal extraction. However, the activity of natural microbial communities is often unstable due to environmental influences.Objective This study constructed a synthetic microbial community comprising six acidophilic (mesophilic or thermophilic) bacterial strains (Acidiphilium cryptum S30H60, Acidithiobacillus caldus SM-1, Acidithiobacillus ferrooxidans KD-1, Alicyclobacillus ferrooxydans TC-34, Leptospirillum ferriphilum T30-1, and Sulfobacillus thermosulfidooxidans T09-1) and one acidophilic thermophilic archaeon (Ferroplasma thermophilum Ferro). The study aimed to investigate the dynamic changes in community structure and function during the bioleaching of pyrite and chalcopyrite at different temperatures.Methods Community dynamics were monitored by qPCR, and the physicochemical parameters of the leaching solution and mineral surface morphology were analyzed. The data were analyzed to reveal the succession patterns of the microbial community and its impact on leaching efficiency under varying temperatures and mineral types.Results The leaching efficiency at 42 ℃ was significantly higher than that at 30 ℃. The microbial community was dominated by S. thermosulfidooxidans and L. ferriphilum. Certain microbial species exhibited distinct preferences for leaching different sulfide minerals.Conclusion Temperature and mineral type significantly influenced the structure and function of the synthetic microbial community. The synergistic dominance of thermophiles at high temperatures enhanced the leaching efficiency. These findings provide a basis for targeted optimization of bioleaching processes.

Abstract:Background Microbial cell-wall membrane structure (CWMS) plays a vital role in microbial-induced mineralization and bioleaching.Objective To reveal the interaction mechanism of Bacillus thuringiensis (JT) CWMS with the albite surface and elucidate the impact during decomposition of silicate minerals.Methods The components of JT CWMS were identified through LC-MS, FTIR, and XPS. The surface wettability, charge, and structure of albite before and after interaction with the CWMS were explored by contact angle measurements, potential analysis, and FTIR and XPS, respectively. DFT calculations were employed to study the adsorption configurations, density of states, charge density, and Mulliken population of small molecular fragments of JT CWMS on the albite surface.Results The JT CWMS contained N-acetylglucosamine (NAG), N-acetylmuramic acid (NAM), phosphoric acid (PA), glycerol, glycine (Gly), D-glutamic acid (D-Glu), and D-alanine (D-Ala). The adsorption strength followed the trend of NAM>PA>glycerol>NAG>D-Glu>Gly>D-Ala. The hydroxyl H of NAG, NAM, PA, and glycerol formed covalent bonds with the O atoms on the albite surface. The amino H of Gly and D-Glu interacted with the O atoms on the surface through hydrogen bonding. The hydroxyl O of NAG, NAM, and glycerol, the carbonyl O of NAM, the double-bond O atom of PA, and the carboxyl O of Gly, D-Glu, and D-Ala formed coordination bonds with the Al atoms on the surface. Additionally, hydrogen bonds existed between the surface silanol groups and oxygen-containing groups of NAG, NAM and glycerol, as well as phosphate groups. The interaction induced the elongation of Si-O and Al-O bonds in the albite unit cell, resulting in a loosened framework structure and the subsequent release of Na⁺. Concurrently, the surface structure, wettability, and charge of albite were altered. Specifically, the hydroxyl and carbonyl groups of NAM preferentially interacted with the albite surface, serving as anchor points for the interaction of the remaining components of the CWMS.Conclusion This study clarifies the interaction between JT CWMS and albite, providing a theoretical basis for microbial promotion of silicate mineral decomposition.

Abstract:Background The Bacillus safensis strain ST7, isolated from the soil of manganese mine pits, exhibits efficient manganese adaptability. A large number of genes are involved in the response to manganese stress, of which the gene encoding the transcription factor stage 0 sporulation protein A (Spo0A) is upregulated and may be involved in regulating the manganese stress response process of B. safensis.Objective To investigate the regulatory role of spo0A in B. safensis ST7 to manganese stress.Methods Genomic DNA was extracted from B. safensis ST7. The gene spo0A was amplified via PCR, cloned into the pSWE-Topo Zero Vector, and then sequenced. The spo0A-deleted mutant (Δspo0A) was constructed via the homologous recombination single-exchange method. The biomass, motility, biofilm formation, manganese oxidation, and sporulation were compared between the wild-type strain and the mutant strain. Additionally, RT-qPCR was performed to compare the expression levels of the sporulation-related gene ctc, quorum sensing-related gene ricR, stress regulation-related gene yodB, and cell surface structure-related gene ywcE.Results The coding sequence of spo0A in B. safensis ST7 was 804 bp in length, encoding 267 amino acid residues, without signal peptide or transmembrane domain. In Δspo0A, the partial sequence of spo0A was replaced by the kanamycin resistance gene, which resulted in the inactivation of spo0A. Under manganese stress, Δspo0A exhibited inhibited growth and reduced biomass. Compared with the wild-type strain, Δspo0A showed the decreases of 13.5%, 85.6%, 18.4%, and 93.36% in motility, biofilm formation, manganese oxidation, and sporulation, respectively. Moreover, the mutant presented down-regulated expression levels of functional genes such as ctc and ricR.Conclusion The gene spo0A can promote flagellar synthesis and motility, enhance the growth and biofilm formation, and regulate the manganese oxidation and sporulation of B. safensis.

Abstract:Background The study of antagonistic microorganisms in the rhizosphere of tobacco-growing regions is crucial for controlling tobacco root rot, ensuring the high quality and high yield of flue-cured tobacco, and promoting sustainable, environmentally friendly production. Objective To screen efficient antagonistic strains against tobacco root rot from the rhizosphere soil of flue-cured tobacco in Chengjiang, Yunnan and investigate their biocontrol efficacy and underlying mechanisms, thus providing a scientific basis for the sustainable control of tobacco root rot in Yunnan. Methods The gradient dilution plate coating method and dual-culture plate assay were employed for strain screening. The isolated antagonistic strains were identified by 16S rRNA gene/ITS sequencing combined with whole-genome analysis. Pot experiments and field trials were conducted to evaluate the biocontrol efficacy of these strains, as well as their effects on the agronomic traits, economic traits, and soil nutrient content of flue-cured tobacco. Furthermore, enzymatic activity assays and metabolomic analyses were performed to elucidate the antagonistic mechanisms. Results Two efficient antagonistic strains were isolated from the rhizosphere soil of flue-cured tobacco in Chengjiang, Yunnan. They were identified as Brevibacillus laterosporus Lc-310 and Talaromyces pseudofuniculosus Dz-1118, which showed the inhibition rates of 71.6% and 72.4%, respectively, against tobacco root rot pathogens in the dual-culture plate assay. Pot experiments demonstrated that the biocontrol efficacy of B. laterosporus Lc-310 and T. pseudofuniculosus Dz-1118 against tobacco root rot was 66.7% and 62.5%, respectively, with the abundance of pathogenic fungi in the rhizosphere soil significantly reducing by 78.7% and 80.7% compared with that in the control group. Field trials further confirmed the biocontrol effects. Compared with the control group, the application of Lc-310 and Dz-1118 decreased the incidence rate of tobacco root rot by 26.5% and 25.1%, reduced the disease index by 54.4% and 51.5%, increased the yield of flue-cured tobacco by 13.1% and 13.8%, and raised the output value by 21.1% and 23.9%, respectively. Moreover, the two strains outperformed a commercially available microbial agent, lowering the disease incidence rate by 10.8% and 9.4%, reducing the disease index by 33.8% and 29.6%, and increasing the yield and output value of flue-cured tobacco. Additionally, the application of Lc-310 and Dz-1118 significantly increased the content of available potassium in the soil by 24.2% and 28.9%, respectively. Regression analysis indicated a significant negative correlation between soil available potassium content and disease index. Both strains secreted cell wall-degrading enzymes, including chitinase and β-1,3-glucanase and produced various antimicrobial metabolites such as surfactin and validamycin A, which directly inhibited the growth and infection of pathogenic fungi. Conclusion B. laterosporus Lc-310 and T. pseudofuniculosus Dz-1118 demonstrate remarkable biocontrol efficacy against tobacco root rot, along with plant growth-promoting effects. Their overall control efficiency surpasses that of the commercial microbial agent used in this study. The biocontrol mechanisms of these two strains involve a synergistic pathway, including direct inhibition of pathogens and indirect enhancement of plant resistance through potassium solubilization. Therefore, the two strains hold great potential for being developed as microbial agents for environmentally friendly biocontrol.

Abstract:Background Befinicial bacteria colonizing the soil of plant roots often exhibit plant growth-promoting properties such as nitrogen fixation, phosphorus solubilization, potassium solubilization, and indole-3-acetic acid (IAA) synthesis, playing an important role in the growth and environmental adaptation of plants. However, the isolation and identification of plant growth-promoting bacteria from the root soil of Oxytropis and the plant growth-promoting effects of these bacteria have been rarely reported.Objective We compared the species of plant growth-promoting bacteria and their plant growth-promoting effects between toxic and non-toxic plants of Oxytropis, aiming to provide theoretical guidance for deciphering the mechanisms of Oxytropis plant-soil bacteria interactions and the ecological management of toxic ones.Methods We employed the culture method to isolate bacteria from the root soil of toxic species O. glacialis, O. falcata, and O. sericopetala as well as non-toxic ones O. microphylla and O. biflora and identified the isolates. Subsequently, we measured the plant growth-promoting properties such as nitrogen fixation, phosphorus solubilization, potassium solubilization, and IAA production of the isolates in vitro. A pot experiment was carried out to verify the growth-promoting effects of different bacterial strains on the seedlings.Results A total of eight strains with the capacity of nitrogen fixation, phosphorus solubilization, and IAA production were screened from 361 isoalted strains, belonging to seven genera (Bacillus, Paeniglutamicibacter, Staphylococcus, Paenarthrobacter, Arthrobacter, Phyllobacterium, and Microbacterium). Plant growth-promoting bacterial strains OgRZ1, OfRZ1, and OsRS1 isolated from the root soil of toxic O. glacialis, O. falcata, and O. sericopetala had stronger phosphorus-solubilizing and IAA-producing capacity than other strains isolated from non-toxic O. microphylla. In the pot experiment, exogenous inoculation of six plant growth-promoting strains significantly increased the root length, aboveground fresh weight, belowground fresh weight, and total fresh weight of O. falcata seedlings by 9.1%-108.8%, 13.8%-197.7%, 28.3%-215.0%, and 18.1%-202.3%, respectively. In addition, inoculation of OgRZ1, OfRZ1, OsRS1, and OsBulk2 significantly increased the seedling height by 22.8%-43.4%. However, the inoculation of these strains had weak impact on swainsonine content in the aboveground part of O. falcata seedlings, and only the inoculation of OmRZ1 and OsBulk1 significantly increased and decreased the swainsonine content, respectively. Furthermore, the effects of plant growth-promoting bacteria on the growth and swainsonine content were positively linked with the IAA production in vitro.Conclusion We isolated eight plant growth-promoting bacterial strains capable of fixing nitrogen, solubilizing phosphorus, and producing IAA from the root soil of toxic and non-toxic plants of Oxytropis. The inoculation of these strains had strong promoting effects on aboveground and belowground growth of O. falcata seedlings but had weak effects on swainsonine content. The effects of these strains were related with the growth-promoting properties such as IAA production. Our study fills the knowledge gap of whether there are differences in the species of plant growth-promoting bacteria in root soil between toxic and non-toxic plants of Oxytropis as well as their growth-promoting effects on seedlings, thereby laying a foundation for further ecological management of toxic Oxytropis plants.

Abstract:Background The long-term excessive application of chemical fertilizer has led to problems such as soil quality degradation and diminishing marginal benefits of fertilizer. Organic fertilizer, rich in soil nutrients and capable of introducing beneficial microorganisms, plays a significant role in improving soil quality and enhancing crop yields.Objective To investigate the effects of partially substituting chemical fertilizer with organic fertilizer on the soil microbial community at different growth stages of maize, providing technical support for the rational application of organic fertilizer.Methods Three treatments: no fertilization (CK), conventional chemical fertilization (CF), and partial substitution of chemical fertilizer with organic fertilizer (OF), were established. Systematic analyses were conducted on changes in soil physicochemical properties and microbial community structure at the seedling and harvest stages.Results Compared with the CF treatment, the OF treatment significantly increased the plant height, stem diameter, and theoretical yield of maize by 10.23%, 17.06%, and 10.32%, respectively. At the seedling stage, the OF treatment significantly increased soil organic matter, alkali-hydrolyzable nitrogen, available phosphorus content, as well as sucrase and urease activities, which indicated effective promotion of soil nutrient supply and carbon-nitrogen transformation during this stage. At the harvest stage, the OF treatment showed no significant difference in soil alkali-hydrolyzable nitrogen or available phosphorus content from the CF treatment but remained significantly higher soil organic matter. Microbial community analysis revealed that the OF treatment significantly enriched beneficial bacteria such as Lysobacter and beneficial fungi such as Podospora and Chaetomium with both plant growth-promoting and antagonistic functions, while reducing the abundance of the pathogenic fungi Paraphoma. Co-occurrence network analysis indicated that the OF treatment significantly enhanced the complexity and stability of the bacterial network at the seedling stage, while this effect weakened at the harvest stage.Conclusion In summary, the partial substitution of chemical fertilizer with organic fertilizer optimizes soil nutrient availability and microbial community structure during the seedling stage, thereby promoting maize growth and increasing the yield. However, such effects diminish at the harvest stage. These findings provide a theoretical basis for reducing chemical fertilizer use while maintaining efficiency and for implementing precision fertilization.

Abstract:Background Ralstonia solanacearum is a soil-borne pathogenic bacterium that causes tomato bacterial wilt and is extremely destructive.Objective To investigate the antimicrobial activity and mechanism of tea tree essential oil against R. solanacearum, thereby providing a reference for the development of plant-derived biological antimicrobial agents.Methods Tea tree essential oil was extracted by steam distillation, and its composition was analyzed by gas chromatography-mass spectrometry (GC-MS) and identified through NIST library comparison. The antimicrobial effects and underlying mechanism of tea tree essential oil against R. solanacearum were investigated by determining the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), effects on the bacterial growth, and fumigation bactericidal activity, as well as measuring its effects on bacterial DNA, proteins, viability, membrane potential, reactive oxygen species (ROS), and biofilm formation.Results The predominant component of tea tree essential oil was terpinen-4-ol (39.83%), and its MIC and MBC against R. solanacearum were 8 mg/mL and 16 mg/mL, respectively. The fumigation test showed that 208.4 mg/L of tea tree essential oil could completely eliminate R. solanacearum. The extracellular DNA concentration increased from 0.10 μg/mL to 0.16 μg/mL, the protein concentration rose from 3.5 mg/mL to 6.4 mg/mL, and the fluorescence intensity of membrane potential decreased sharply from 1 335.6 a.u. to 177.2 a.u., accompanied by a decline in bacterial viability. These results indicated that tea tree essential oil could damage the cell membrane and increase its permeability. Fluorescence spectroscopy analysis and confocal laser scanning microscopy observations demonstrated that tea tree essential oil could target bacterial DNA and reduce its content. Additionally, the fluorescence intensity of ROS increased from 638.5 a.u. to 1 595.4 a.u., suggesting that excessive ROS accumulation induced bacterial damage. Fluorescence spectroscopy and molecular docking analysis showed that tea tree essential oil could bind to proteins, and that terpinen-4-ol could act on the target protein DNA gyrase subunit B, dihydrofolate reductase and peptide deformylase, thereby exerting antimicrobial effects.Conclusion Tea tree essential oil exerts an antimicrobial effect on R. solanacearum through multiple mechanisms, providing a theoretical basis for the development of plant-derived biological antimicrobial agents to prevent and control tomato bacterial wilt.

Abstract:Background Rice sheath blight, caused by Rhizoctonia solani, is a major disease limiting high rice yields. Long-term sole reliance on chemical control easily exacerbates pathogen resistance and poses environmental risks. Although biological control agents (BCAs) are environmentally friendly, their application is restricted by weak colonization ability and unstable control efficacy. Thus, the combined application of BCAs and chemical fungicides is a promising development direction.Objective To isolate and identify endophytic bacteria with high biocontrol potential against rice sheath blight, clarify their taxonomic status and molecular characteristics, explore the effects of tebuconazole on bacterial growth and biofilm formation, and evaluate the control efficacy of the strain combined with tebuconazole against rice sheath blight, thereby providing new bacterial resources and molecular mechanism support for the control of rice sheath blight with reduced chemical use and enhanced efficacy.Methods Endophytic bacteria were isolated from healthy rice sheaths inoculated with R. solani. Efficient biocontrol strains were obtained through dual screening based on plate antifungal activity and sclerotial germination inhibition. The taxonomic status of the strain was identified by morphological, physiological, and biochemical characterization, as well as sequence analysis of 16S rRNA and gyrA genes. Whole-genome sequencing was performed to analyze genomic features. In vitro experiments were conducted to investigate the effects of tebuconazole at different concentrations on bacterial biofilm formation (quantified by OD₅₈₁ absorbance) and growth (quantified by OD₆₀₀ absorbance). Pot experiments were carried out to evaluate the actual control efficacy of the combined application of the strain and tebuconazole, which was compared with the theoretical efficacy calculated by the Colby’s method.Results An efficient biocontrol strain, designated SAU-22, was isolated and identified as Bacillus velezensis. It exhibited high gene annotation efficiency, carrying 12 secondary metabolite biosynthetic gene clusters, 11 resistance genes, and 2 genes associated with biofilm formation. The plate inhibition rate of SAU-22 against R. solani reached 70.23%, and the sclerotial germination rate of the pathogen decreased to 13.33% after treatment with strain SAU-22 fermentation broth. Tebuconazole at concentrations of 1.56-6.25 mg/L promoted the biofilm formation and growth of strain SAU-22, while that at concentrations ≥25 mg/L significantly inhibited these traits. Pot experiment results showed that the actual control efficacy of the combination of 80 mg/L tebuconazole and strain SAU-22 against rice sheath blight reached 100%, which was significantly higher than the theoretical efficacy (82.56%) calculated by the Colby’s method and the efficacy of other combination groups.Conclusion Strain SAU-22 is a promising commercial biocontrol strain with high efficacy against rice sheath blight. The abundant antifungal metabolite biosynthetic gene clusters in its genome provide a molecular basis for its biocontrol function. Low concentrations of tebuconazole can promote the growth and biofilm formation of strain SAU-22, and their combination exhibits a significant synergistic control effect, achieving the goal of reduced chemical use and enhanced efficacy. This study provides new bacterial resources and a theoretical basis for the construction of a green control system for rice sheath blight, as well as practical reference for the synergistic application of BCAs and chemical fungicides.

Abstract:Background The leaf blight of Schisandra sphenanthera in Yunxi County, Hubei province has seriously affected the healthy development of the local S. sphenanthera industry. Identifying the pathogens of this disease and screening effective control agents have become urgent tasks. Objective To clarify the population structures, pathogenicity, and biological characteristics of the pathogens causing leaf blight, and to screen effective fungicides, thus providing a theoretical basis for understanding pathogen diversity and developing effective control measures against S. sphenanthera leaf blight in Yunxi county, Hubei provine. Methods Leaf blight samples were collected, and a phylogenetic tree was constructed based on rDNA-ITS sequence analysis to clarify the taxonomic positions of pathogen populations. Five representative strains were selected for further characterization of pathogenicity and biological characteristics, and the indoor inhibitory activities of various fungicides against these strains were determined. Results The 28 pathogenic strains were clustered into four phylogenetic clades, with Alternaria sp. being the dominant population (89.29%), followed by Didymella sp. (10.71%). Significant differences in pathogenicity and biological characteristics were observed among the five representative strains. The optimal pH value for their mycelial growth ranged from 4.0 to 11.0. The optimal nitrogen source was sodium nitrate, and the optimal carbon source was glucose or soluble starch. The suitable growth temperature was distributed between 20 ℃ and 30 ℃. The strains could tolerate salt with the concentration ranging from 2.50 g/L to 20.00 g/L, and the minimum lethal temperatures were between 50 ℃ and 55 ℃. In vitro antifungal activity assays indicated that difenoconazole, prochloraz, tebuconazole·prochloraz, and benzoyl·prochloraz exhibited strong inhibitory effects against all five pathogenic strains. Conclusion The findings of this study provide the first clarification of the population diversity, pathogenicity variation, and biological characteristics of the pathogens causing S. sphenanthera leaf blight, along with the identification of effective fungicides, thereby establishing a theoretical foundation for the development of targeted disease management strategies.

Abstract:Background Bacillus velezensis Ba-0321 obtained previously is a biocontrol strain with plant growth-promoting and disease-preventing effects, capable of producing a variety of antimicrobial substances.Objective This study screened novel broad-spectrum and effective antimicrobial proteins, analyzed their antimicrobial mechanisms, and determined their control efficacy against tobacco diseases, aiming to lay a foundation for developing new antimicrobial agents.Methods Extracellular crude proteins from Ba-0321 were isolated through ammonium sulfate precipitation at varying saturation levels, followed by centrifugation and dialysis. The Oxford cup method was employed to assess the antimicrobial activity, and the protein stability was evaluated under different temperature and pH conditions. The antimicrobial components within the crude protein extracts were further characterized by LC-MS/MS. Histochemical staining was combined with qPCR analysis of disease resistance-related genes to reveal the mechanisms of tobacco resistance induced by the crude proteins. Inoculation assays were conducted to validate the efficacy of these proteins against Fusarium root rot and brown spot disease in tobacco.Results When ammonium sulfate saturation was 40%, the extracellular crude proteins precipitated from the fermentation supernatant of Ba-0321 exhibited the strongest antifungal activity against Fusarium oxysporum, with an inhibition rate of 49.16%. The proteins remained stable at 40-90 ℃ and pH 7.0-11.0. Moreover, they exhibited inhibitory activities against other tobacco pathogenic fungi including Fusarium solani, Phytophthora nicotianae, Rhizoctonia solani, Alternaria alternata, and Diaporthe. LC-MS/MS analysis identified multiple antimicrobial proteins in the extracellular crude protein fraction of Ba-0321, which included β-glucanase, chitosanase, α-amylase, subtilisin, serine protease, chitin-binding protein, and the antimicrobial peptide LCI, along with seven unidentified proteins. The crude proteins triggered hypersensitive response (HR) and hydrogen peroxide (H₂O₂) accumulation in tobacco, activated the expression of key genes (including NtNPR1, NtPR1a, NtEIN2, NtEIN3, NtJAR1, and NtPDF1.2) involved in salicylic acid (SA), ethylene (ET), and jasmonic acid (JA) signaling pathways, thereby inducing systemic disease resistance in tobacco. The control efficacies of extracellular crude proteins from Ba-0321 against the root rot caused by F. oxysporum and the brown spot disease caused by A. alternata in tobacco reached 42.88% and 68.87%, respectively.Conclusion The extracellular crude proteins of strain Ba-0321 can be applied as biopesticides or plant immunity inducers for the green control of tobacco diseases.

Abstract:Background Soil salinization is a major abiotic stress limiting agricultural production. Utilizing beneficial rhizosphere microorganisms to improve crop salt tolerance has been recognized as an effective and sustainable strategy.Objective To isolate salt-tolerant bacteria from the rhizosphere soil of Suaeda salsa L., investigate their effects on barley salt tolerance, and decipher the underlying physiological and molecular mechanisms.Methods A bacterial strain STPM1 (salt-tolerant Priestia megaterium 1) isolated from saline-alkali soil was identified through morphological characterization and molecular analysis. Its tolerance to salt and alkali, as well as its plant growth-promoting traits, were evaluated. The effects of STPM1 inoculation on barley under NaCl stress were comprehensively assessed in terms of seed germination, seedling growth, physiological and biochemical properties, and expression of salt stress-responsive genes.Results Strain STPM1 was identified as P. megaterium, a Gram-positive bacterium with capsule and capable of forming spores and growing in the media containing up to 12% NaCl and within the range of pH 5.0-9.0. The strain exhibited multiple plant growth-promoting traits, including nitrogen fixation, phosphate and potassium solubilization, and secretion of phytohormones such as indole-3-acetic acid (IAA) and abscisic acid (ABA). Under salt stress, inoculation with STPM1 significantly improved the seed germination, promoted seedling growth, enhanced antioxidant enzyme activities, increased the accumulation of proline and ABA, and regulated the transcriptional levels of several salt stress-responsive genes in barley.Conclusion The salt-tolerant rhizobacterium STPM1 enhances the salt tolerance of barley by activating the antioxidant defense system and the ABA signaling pathway, demonstrating the potential as a microbial inoculant for improving crop performance in saline-alkali soils.

Abstract:Background Rhizospheric and plant-derived endophytic bacteria harboring 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity play a critical role in facilitating host plant adaptation to environmental stress by modulating key physiological and developmental processes.Objective To isolate cultivable bacteria from the rhizosphere and various tissues of Dongxiang wild rice (Oryza rufipogon), characterize their taxonomic composition and diversity, evaluate their plant growth-promoting traits, and preliminarily investigate the potential link between ACC deaminase-active strains and the stress tolerance of Dongxiang wild rice.Methods In this study, 118 strains of culturable rhizosphere and endophytic bacteria were isolated from Dongxiang wild rice. The taxonomic positions of these strains were determined through 16S rRNA gene sequence analysis. The strains exhibiting ACC deaminase activity were screened, and quantitative assays were conducted to measure their ACC deaminase activity and other plant growth-promoting traits.Results The 118 strains of rhizosphere and endophytic bacteria belonged to 22 genera, 16 families, 13 orders, seven classes of four phyla, with the dominant genera being Bacillus and Pantoea. A total of 29 strains with ACC deaminase activity were identified. Four strains exhibited phosphorus-solubilizing ability; 20 strains produced indole-3-acetic acid (IAA); and five strains were capable of producing siderophores.Conclusion The findings indicate that Dongxiang wild rice exhibits rich bacterial diversity in various tissues and rhizosphere soil. Moreover, there are a high proportion of strains with ACC deaminase activity and the isolated strains have multiple plant growth-promoting traits. These features position the strains as a promising new source of beneficial microbes for enhancing soil fertility and improving rice stress tolerance.

Abstract:Background Fire blight, caused by Erwinia amylovora, is a devastating bacterial disease affecting the global fruit plant industry of Rosaceae.Objective Explored the inhibitory effect of 2-phenylacrylic acid at subinhibitory concentrations against the quorum sensing of E. amylovora, aiming to underpin the development of novel control agents.Methods The minimum inhibitory concentration (MIC) of 2-phenylacrylic acid against strain Ea-213 was determined. The effects of 2-phenylacrylic acid at subinhibitory concentrations on the biofilm formation, extracellular polysaccharide production, motility, and activities of various cell wall-degrading enzymes were evaluated via crystal violet staining and the anthrone-sulfuric acid assayResults The MIC of 2-phenylacrylic acid was determined to be 1 mg/mL against strain Ea-213 and 0.5 mg/mL against the Gram-negative biosensor strain CV026. At subinhibitory concentrations, 2-phenylacrylic acid did not affect the growth of strain Ea-213 but exhibited pronounced quorum-quenching activity against CV026. At 1/2 MIC, 2-phenylacrylic acid significantly inhibited the biofilm formation, extracellular polysaccharide production, and motility of strain Ea-213. Furthermore, the compound markedly suppressed the activities of cellulase, xylanase, pectin lyase, and polygalacturonase secreted by E. amylovora. Particularly, 2-phenylacrylic acid exerted the most significant inhibitory effect on polygalacturonase, with an inhibition rate reaching 53.8% at 1/2 MIC.Conclusion 2-Phenylacrylic acid can effectively attenuate multiple key virulence factors of E. amylovora by interfering with its quorum sensing system, demonstrating considerable potential as a novel biocontrol agent against fire blight.

Abstract:Background Forest-dwelling cockroaches play an important role in maintaining the balance and stability of forest ecosystems. Their habitats are rich in diverse entomopathogenic fungi, serving as a natural reservoir for exploring cockroach-specific biocontrol agents.Objective To investigate the diversity of entomopathogenic fungi associated with Asian forest-dwelling cockroaches, clarify their taxonomic status, and evaluate their pathogenic potential against the urban pest Periplaneta americana, thereby providing novel fungal resources for the management of urban cockroach populations with aggravating insecticide resistance.Methods Infected cockroach specimens were collected from forests in southwestern China and the Indochinese Peninsula, followed by fungal isolation and purification. Taxonomic identification was conducted based on multi-gene phylogenetic analyses combined with microscopic morphological observations. The virulence of the isolates was evaluated by conidial germination assays and laboratory bioassays.Results A total of five fungal taxa were identified from forest-dwelling cockroaches, including one new species, Beauveria laosensis, and four known species: B. bassiana, Metarhizium blattodeae, M. pemphigi, and Ophiocordyceps blattae. Pathogenicity assays demonstrated that only Beauveria species were capable of infecting P. americana, with B. bassiana causing the highest mortality, whereas the newly described B. laosensis showed comparatively lower virulence.Conclusion This study expands current knowledge of the diversity of cockroach-associated entomopathogenic fungi, provides a formal description and illustration of the new species B. laosensis, and highlights the ecological significance of forest-dwelling cockroaches as important reservoirs of entomopathogenic fungi. These findings provide valuable fungal resources and a theoretical basis for the development of specialized biocontrol agents targeting P. americana.

Abstract:Background The interactions between probiotics and core taxa such as Bacteroides in the gut microbiota are crucial prerequisites for their functional efficacy. However, the specific microscopic interaction mechanisms and species-specific metabolic cross-feeding patterns remain to be elucidated.Objective This study established in vitro co-culture models of Lactiplantibacillus plantarum P-8 with three Bacteroides strains (B. xylanisolvens, B. uniformis, and B. eggerthii) to analyze community evolution and metabolic profiles across different time points, aiming to elucidate the species-specific interactions and cross-species metabolic complementarity.Methods Dual-strain co-culture systems consisting of P-8 and either B. xylanisolvens, B. uniformis, or B. eggerthii were constructed with the modified GAM medium. OD₆₀₀ values were recorded at the time points of 0, 12, 24, and 48 h for establishment of growth curves. Metagenomic and non-targeted metabolomic analyses were performed to characterize the community structures and metabolic interaction features at the time points of 24 h and 48 h.Results P-8 established stable coexistence with all the three Bacteroides strains, although the interaction patterns exhibited significant species-specific differences. Specifically, the co-culture groups involving B. uniformis and B. eggerthii demonstrated a pronounced over-yielding effect, with the total biomass at 48 h being significantly higher than that of either monoculture (P<0.05). In the co-culture system with B. xylanisolvens, the Bacteroides strain remained the dominant taxon throughout cultivation, accounting for more than 90% of the community abundance, and the metabolic profile of the co-culture closely resembled that of the B. xylanisolvens monoculture, characterized by the sustained accumulation of organic acids such as succinate. Conversely, in the co-culture of P-8 with B. uniformis or B. eggerthii, distinct metabolic transition was observed: phenylalanine was consumed within 24 h and subsequently converted into phenyllactic acid within 48 h. This temporal metabolic evolution highlighted the existence of cross-species metabolic complementarity between P-8 and specific Bacteroides species.Conclusion P-8 maintains robust symbiotic relationships with Bacteroides strains. However, its functional output is highly dependent on the co-existing Bacteroides species. This metabolic complementarity enhances the synthesis of secondary metabolites and provides a theoretical basis for understanding the individualized efficacy and colonization differences of probiotics in diverse gut environments.

Abstract:Background Avian pathogenic Escherichia coli (APEC) is extraintestinal pathogenic E. coli that can invade extraintestinal organs in poultry, causing colibacillosis, which manifests as various symptoms including acute septicemia, peritonitis, perihepatitis, and pericarditis. Currently, the treatment of avian colibacillosis mainly relies on antibiotics. However, the severe multidrug resistance of APEC limits the control of this disease. Moreover, APEC can resist antibiotic killing and evade host immunity through biofilm formation. Therefore, the research on alternative products to antibiotics has become a hot topic. Berberine extracted from Chinese herbal medicines has broad-spectrum antibacterial properties. Thus, exploring the effects and regulatory mechanisms of berberine on the biofilm formation of APEC is of great significance for the prevention and control of avian colibacillosis with berberine as an alternative of antibiotics.Objective This study investigated the effects of berberine on the biofilm formation of APEC and deciphered the underlying mechanisms, aiming to provide theoretical references for the prevention and control of avian colibacillosis.Methods Berberine was applied to APEC, and its minimum inhibitory concentration against APEC was determined. Furthermore, the effects of berberine on APEC were characterized in terms of biofilm formation, outer and inner membrane permeability, intracellular ATP levels, intracellular reactive oxygen species generation, and transcription levels of biofilm formation-related genes.Results At certain concentrations, berberine did not affect the growth, membrane permeability, intracellular ATP levels, or reactive oxygen species levels of APEC. However, it reduced the biofilm formation of APEC. Scanning electron microscopy showed that berberine resulted in sparse bacterial cells and reduced interbacterial adhesion in APEC biofilms. Confocal laser scanning microscopy revealed that berberine led to sparse distribution of bacteria in the horizontal plane, thinning, and an overall loose structure of the biofilm. Real-time quantitative PCR analysis showed that berberine reduced the transcription levels of biofilm formation-related genes (csgA and wza) in APEC.Conclusion Berberine can affect the biofilm formation of APEC by influencing the transcription of biofilm formation-related genes, which is manifested as the destruction of the structural integrity of biofilms.

Abstract:Background Aeromonas veronii has attracted considerable attention as a common pathogen in aquaculture, with its pathogenicity highly associated with the type III secretion system (T3SS), although the underlying mechanisms remain incompletely understood. Objective To provide a theoretical basis for developing an ascV-targeted attenuated live vaccine candidate. We investigated the role of ascV, a critical component of the T3SS, in the pathogenicity of A. veronii strain AV1, which was isolated from Channa argus. An ascV-deleted mutant was constructed and characterized in terms of biological functions, adhesion and invasion to EPC cells, and overall pathogenicity. Methods A suicide plasmid pRE112-Δ ascV was constructed via molecular cloning for homologous recombination. The recombinant plasmid was transformed into Escherichia coli S17-1λpir and subsequently introduced into the wild-type AV1 strain through conjugational transfer. After two rounds of homologous recombination screening, the ascV-deleted mutant AV1-Δ ascV was successfully obtained via the selection media containing chloramphenicol and sucrose. The mutant was verified by colony PCR and sequencing. The biological characteristics, adhesion and invasion to epithelioma papulosum cyprinid cell line (EPC), as well as the pathogenicity of both wild-type and mutant strains, were systematically evaluated. Results Growth curve and motility assays revealed no significant differences in growth rates between the mutant and wild-type strains, suggesting that the deletion of ascV did not affect basic growth metabolism. In terms of extracellular enzyme and hemolytic activities, no significant differences were observed between the wild-type and the mutant strains in protease, phospholipase, amylase, lipase, and β-hemolytic activities. However, biofilm formation was significantly reduced in the mutant compared with that in the wild-type strain ( P<0.05), indicating that ascV played a critical role in regulating biofilm formation. The adhesion and invasion of AV1-Δ ascV to EPC cells decreased ( P<0.05). The LD ₅₀ values of AV1-Δ ascV against C. argus and Carassius auratus were significantly higher than those of the wild-type strain, indicating the significantly attenuated virulence of the mutant. AV1-Δ ascV alleviated gill damage and reduced mortality in C. argus and C. auratus after infection compared with the wild-type strain. Conclusion This study successfully constructed an ascV-deleted mutant of A. veronii and demonstrated that while the gene deletion did not affect fundamental growth, motility, or extracellular enzyme activities in the mutant, it significantly impaired biofilm formation and reduced the adhesion and invasion to EPC cells, simultaneously lowering the pathogenicity to C. argus and C. auratus.

Abstract:Background Bacterial enteritis in chicken is a major disease threatening the poultry industry. The enhancing antibiotic resistance of its main pathogens, such as Escherichia coli and Salmonella, has reduced the efficacy of antibiotic treatments. Colistin sulfate is a key drug for treating Gram-negative bacterial infections, while the emergence of resistance limits its application. Eugenol, as a natural plant extract, possesses antibacterial activity. Investigating the synergistic effects and mechanisms of the combined use of colistin sulfate and eugenol is essential for developing new antibacterial strategies and combating antibiotic-resistant bacterial infections.Objective To predict the synergistic targets and pathways of colistin sulfate combined with eugenol against the main pathogens (E. coli and Salmonella) of bacterial enteritis in chicken by network pharmacology, and validate their synergistic antibacterial effect, bactericidal dynamics, and ability to delay the development of bacterial resistance through in vitro experiments.Methods Potential common targets and signaling pathways of colistin sulfate and eugenol against E. coli and Salmonella were screened via network pharmacology. A protein-protein interaction (PPI) network was constructed, followed by Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. The minimum inhibitory concentration (MIC) was determined by the microdilution broth method. The fractional inhibitory concentration index (FICI) was calculated by the checkerboard assay, and the bactericidal dynamics was assessed via the time-kill curve. The development of bacterial resistance was investigated through a 30-day sub-MIC passage assay.Results Network pharmacology screening identified 145 potential synergistic targets for the combined treatment of bacterial enteritis in chicken, with core PPI network targets including MAPK3, FoxO1, and CARD9. GO and KEGG enrichment analyses indicated that the synergistic mechanisms involved pathways such as the Toll-like receptor (TLR)/NOD-like receptor (NLR) signaling pathway, apoptosis/necroptosis, and the FoxO signaling pathway. The in vitro experiments confirmed that the combination produced significant synergistic effects against all the 15 tested strains (FICI≤0.5), reducing the MIC of colistin sulfate by 6.25%-25.00% and the MIC of eugenol by 6.25%-25.00%. The minimum resistance reversal concentration (MRC) test successfully restored the susceptibility of resistant strains to colistin sulfate (MIC≤2 μg/mL). Time-kill curves showed complete bacterial eradication within 4 h by the combination. The 30-day passage assay demonstrated that the increase in resistance in the combination group was only two folds, which was significantly lower than those (16-64 folds) in the monotherapy groups.Conclusion The combination of colistin sulfate and eugenol exerts synergistic antibacterial effects through multiple targets and pathways and effectively delays the development of bacterial resistance. This provides a new strategy for preventing and treating bacterial enteritis in chicken and combating multidrug-resistant bacterial infections.

Abstract:Background Studies have shown that inhibiting human cytochrome P450 8B1 (CYP8B1) can regulate bile acid metabolism and further influence glucose and lipid homeostasis, representing a potential therapeutic strategy for metabolic diseases such as type 2 diabetes, obesity, and non-alcoholic fatty liver disease. However, the development of CYP8B1 inhibitors remains slow, possibly due to the inability of existing small-molecule screening systems targeting CYP8B1 activity to achieve medium-to-high-throughput screening.Objective This study constructed an Escherichia coli system co-expressing human CYP8B1 and cytochrome P450 reductase (CPR) and identified the strains with high catalytic activity of 12α-hydroxylase, aiming to establish a medium-to-high-throughput screening system for CYP8B1 activity.Methods First, on the basis of bicistronic or dual-expression vector strategies, recombinant plasmids co-expressing CYP8B1 and CPR were constructed with the medium-to-high-copy plasmid pET-17b or the high-copy plasmid pRSFDuet-1 as vectors. Subsequently, pGro7/pGro12 (capable of expressing the molecular chaperone proteins groES-groEL) and the constructed co-expression plasmids were successively transformed into different host strains C41(DE3), C43(DE3), BL21-CodonPlus(DE3)-RIPL, Rosetta(DE3), and GSsetta(DE3) to construct different recombinant expression strains. After fermentation of the recombinant strains, 7α-hydroxy-4-cholesten-3-one (7α-HCO) was used as the substrate for CYP8B1, and reverse phase-high performance liquid chromatography (RP-HPLC) was employed to measure the whole-cell catalytic activity. Finally, the optimal reaction conditions and expression system were determined by comparing the activity assay results.Results We successfully constructed a heterologous expression system of human CYP8B1 in E. coli. With the medium-to-high-copy plasmid pET-17b and the high-copy plasmid pRSFDuet-1 as vectors, along with plasmids pGro7 and pGro12 for expressing the molecular chaperone proteins groES-groEL, 12 recombinant expression strains of CYP8B1 were constructed in five host strains: C41(DE3), C43(DE3), BL21-CodonPlus(DE3)-RIPL, Rosetta(DE3), and GSsetta(DE3). By comparing their whole-cell activities, we identified the recombinant strain C43-pRSF-tCYP8B1-tCPR as the optimal expression system for CYP8B1, with the optimal reaction conditions being no polymyxin B added to the reaction system and dimethyl sulfoxide (DMSO) as the solvent for the substrate 7α-HCO.Conclusion The engineered E. coli system enables effective heterologous expression of human CYP8B1 with robust whole-cell catalytic activity.

Abstract:Background Soil salinization and available phosphorus deficiency are among the main factors restricting global agricultural development. In this context, the use of functional microorganisms is considered the most promising green improvement solution due to its environmental friendliness and no secondary pollution.Objective Enterobacter cloacae AZ8 is a strain of bacteria isolated by our research group from Egyptian salt-alkali soil samples, with both salt-alkali tolerance and efficient phosphorus solubilization. This study delved into its genome sequence information and molecular mechanisms of salt-alkali tolerance and efficient phosphorus solubilization, aiming to provide a theoretical basis and bacterial resources for salt-alkali soil remediation.Methods This study used the concentration gradient method to test the salt-alkali tolerance of the bacterial strains. The plate method and molybdenum-antimony colorimetric method were employed to preliminarily evaluate the phosphorus-solubilizing effect of the strain. The strain was identified through morphological observation, physiological and biochemical analysis, and 16S rRNA sequencing. Illumina sequencing and PacBio sequencing were employed to obtain the whole genome of AZ8. Bioinformatics methods were used for genome sequence assembly, gene function annotation, and gene prediction to further explore functional genes and elucidate the mechanisms underlying the salt-alkali tolerance and phosphate solubilization of strain AZ8.Results Strain AZ8 grew well under 7.5% NaCl and pH 10.0. It demonstrated significant phosphorus-solubilizing effect, with the highest solubilizing abilities reaching 395.56 μg/mL for organic phosphorus and 363.93 μg/mL for inorganic phosphorus. Whole genome sequencing confirmed that strain AZ8 as E. cloacae and this strain carried multiple phosphorus solubilization-related genes, such as citrate synthase gene (gltA), malate synthase gene (aceB), and inorganic phosphate transport genes (pstA, pstB, pstC, and pstS). In terms of salt-alkali tolerance, the strain carried two Na⁺/H⁺ antiporters NhaA and NhaB, as well as glycine betaine transport-related genes, such as betA, betB, and betT.Conclusion E. cloacae AZ8 has strong salt-alkali tolerance and efficient phosphorus solubilization. Whole genome analysis further reveals the genetic basis and potential mechanisms at the molecular level, providing theoretical guidance for salt-alkali land remediation and phosphorus activation.

Abstract:Background Lactobacilli, as core probiotics, are the preferred strains for microbial feed additives in animal farming, and the intestinal Lactobacilli resources of simmental calves in China have not been fully explored.Objective To screen the Lactobacilli strains with excellent probiotic properties as potential probiotics from the intestinal tract of simmental calves.Methods The feces samples of 7-day-old calves were collected for the isolation of strains, which were then identified by morphological observation, physiological and biochemical tests, and 16S rRNA gene sequencing. Phenotypic tests were conducted to evaluate the strain’s thermotolerance, acid tolerance, bile salt tolerance, gastrointestinal tolerance, antibacterial effects, antibiotic sensitivity, and haemolytic activity. Illumina and PacBio platforms were used for high-throughput sequencing of the whole genome, and bioinformatics tools were used for gene prediction and functional annotation. The probiotic properties and safety were assessed considering both phenotypes and genes.Results A strain Lacticaseibacillus paracasei KQ NIU was isolated and identified. In vitro tests showed that this strain not only had good thermotolerance, acid tolerance, bile salt tolerance, and gastrointestinal tolerance but also inhibited Escherichia coli, Salmonella, Staphylococcus aureus. It was sensitive to nine antibiotics and non-haemolytic, showing high safety. The whole genome of the strain was 3 145 357 bp in length, with the G+C content of 46.36%, encoding a total of 2 978 genes. A total of 15 rRNAs and 59 tRNAs were predicted. In addition, probiotic genes related to tolerance to heat and cold stress, acids, and bile salts, adhesion, and antioxidant capacity were identified. No antibiotic resistance gene or virulence gene was identified.Conclusion The strain screened in this study has good probiotic properties and safety, and it can be used as a candidate strain for microecological preparations, providing resources and a theoretical basis for the research and application of intestinal probiotics in beef calves.

Abstract:Background The non-structural protein 1 (NS1) of the influenza A virus is a key factor in antagonizing the host’s innate immune response. Its amino acid variations may significantly influence viral replication fitness and immune evasion capabilities.Objective To construct back-mutant viruses at specific sites of NS1 to investigate the effects of these mutations on the replication and transcription processes of the influenza virus across different cell lines.Methods With the A/California/07/2009 (H1N1) strain as the backbone, recombinant viruses carrying R108K, G189D, and double mutation were constructed. The replication kinetics of the mutant strains were compared with the wild-type (WT) virus in MDCK and A549 cell lines. Furthermore, the dynamics of viral vRNA, cRNA, and mRNA were detected by RT-qPCR.Results Sequence analysis revealed that K108R and D189G mutations occurred in the NS1 of H1N1 subtype influenza viruses around the 2009 pandemic. The constructed mutant strains carrying R108K, G189D, and double mutation showed no significant differences in proliferation levels compared with the WT virus in MDCK cells. However, in A549 cells, the replication capacity of the mutant strains was significantly weaker than that of the WT. The G189D mutant exhibited higher levels of vRNA and cRNA than the WT, suggesting that this mutation specifically enhanced the efficiency of viral genome replication. Conversely, R108K and the double mutation primarily promoted mRNA synthesis.Conclusion The effects of back-mutations at positions 108 and 189 of NS1 on viral replication efficiency were host-dependent. Although these mutations enhanced RNA synthesis, the overall yield of the viral life cycle was inhibited within the specific host cell environments tested. This suggests that the evolutionary selection at these sites may be influenced by interactions with specific internal factors in host cells.

Abstract:Background Bacterial resistance poses a serious threat to human and animal health. The formation of biofilms is one of the main reasons for bacterial resistance. Conventional antibacterial drugs have difficulty in penetrating into biofilms and bacteria are prone to developing resistance. In this study, the Mn-Zn-ZIF material loaded with cellulase (CL) and glucose oxidase (GOX) was constructed based on the cascade effect of CL and GOX.Objective To achieve the degradation of extracellular matrix in biofilms and the efficient bactericidal effect of reactive oxygen species (ROS).Methods The CL and GOX-loaded Mn-Zn-ZIF composite material (CL&GOX@Mn-Zn-ZIF) was prepared by a one-pot hydrothermal method and then characterized. The minimum inhibitory concentrations (MICs) and inhibition rates of CL&GOX@Mn-Zn-ZIF against Staphylococcus aureus and Escherichia coli were determined by the serial dilution method. The ability of CL&GOX@Mn-Zn-ZIF to produce ROS and its effect on the integrity of bacterial cell membranes were investigated. The clearance of mature biofilms by CL&GOX@Mn-Zn-ZIF was determined by crystal violet staining, and its cellulose degradation ability was examined.Results CL&GOX@Mn-Zn-ZIF effectively loaded CL and GOX, and its MICs against E. coli and S. aureus were both 160 μg/mL. The treatment with CL&GOX@Mn-Zn-ZIF and β-D-glucose increased the ROS production and promoted the leakage of eDNA from S. aureus and E. coli (P<0.05). In addition, CL&GOX@Mn-Zn-ZIF showed enhanced performance in clearing the mature biofilms of S. aureus and E. coli (P<0.05), and it could significantly degrade cellulose to destroy biofilms.Conclusion CL&GOX@Mn-Zn-ZIF can inhibit growth, disrupt the cell membrane structure, and clear the mature biofilms of E. coli and S. aureus, thus being expected to be a new and effective antibacterial strategy.

Abstract:Background Pseudomonas aeruginosa is a common human opportunistic pathogen characterized by high virulence and pathogenicity. Antibiotic therapy remains the primary approach for controlling P. aeruginosa, while phages represent one of the effective alternative biocontrol strategies to antibiotics.Objective To develop a pH-responsive, gut-targeted phage-based drug delivery system and explore the feasibility of using this delivery system to prevent and treat P. aeruginosa intestinal infections by evaluating its material properties, biological characteristics, and antibacterial efficacy.Methods A virulent phage strain, ws-1, was isolated and screened with P. aeruginosa as the host bacterium. The phage was characterized in terms of the optimal multiplicity of infection, one-step growth curve, and tolerance to temperature and pH. We prepared the delivery system ws-1@LC by encapsulating phage ws-1 with L100-55-catechin (LC). The drug loading capacity and release behavior of ws-1@LC were studied through UV spectroscopy, particle size and zeta potential measurements, and gastrointestinal fluid digestion tests. Additionally, the therapeutic efficacy of ws-1@LC against P. aeruginosa intestinal infections was evaluated by animal experiments.Results Phage ws-1 exhibited strong specificity toward P. aeruginosa, with incubation and bursting periods of 15 min and 40 min, respectively. Its survival rates exceeded 90% across the temperature range from 25 ℃ to 50 ℃ and pH 5.0-9.0. Encapsulating phage ws-1 with LC increased the average particle size from (46.1±3.9) nm to (86.31±1.8) nm and decreased the Zeta potential from (-12.5±1.1) mV to (-15.9±1.1) mV. In addition, ws-1@LC exhibited enhanced tolerance to gastric fluid and released phage ws-1 and catechin in intestinal fluid. Compared with ws-1+LC, ws-1@LC showed reduced the P. aeruginosa count and inflammatory damage in the intestinal tissue of mice.Conclusion The pH-responsive LC encapsulation of phage ws-1 yields the drug delivery system ws-1@LC for targeted intestinal release, which demonstrates effective prevention and treatment against P. aeruginosa intestinal infections. This provides a research foundation for developing novel phage-based formulations.

Abstract:Background Plant growth-promoting microbes (PGPM) play a vital role in enhancing crop productivity, stress tolerance, and agricultural sustainability through a variety of well-documented mechanisms, including facilitation of nutrient assimilation, and production of beneficial metabolites. They are increasingly recognized as a core biotechnology driving the development of green agriculture. Nevertheless, quantitative and systematic analyses in this field remain scarce at present.Objective To elucidate the research hotspots and trends in this field, this study employed bibliometric methods.Methods With the CiteSpace visualization tool, we analyzed PGPM-related publications indexed in the Web of Science Core Collection from 2015 to 2024.Results The analysis reveals that the annual number of publications globally on PGPM has shown a rapid and stable growth trend over the past decade, with countries such as the United States, China, India, and Saudi Arabia playing important roles. King Saud University and the Chinese Academy of Sciences are the top two institutions in terms of publication volume. Current high-frequency keywords in this field include “PGPM-mediated stress resistance mechanisms” “analysis of PGPM communities” and “biofortification”. Research directions are shifting from single-mechanism studies to multi-omics integration, from investigations on individual strains to microbial community interactions, and from yield enhancement toward crop quality improvement—reflecting a transition from fundamental research to applied innovation. In addition, analysis of publication output and citation performance showed that China has consistently ranked among the top countries in annual publication volume. The median citation frequency of Chinese literature is 23, which is higher than the world median level. This demonstrates China’s solid research foundation and growing academic influence in the PGPM field. However, the relatively low betweenness centrality of Chinese publications suggests that their originality and international academic impact remain to be strengthened.Conclusion To further advance the field, research institutions should build upon existing strengths and focus on: (1) enhancing original innovation through discovering novel microbial resources and studying their mechanisms (2) deepening international collaboration and increasing global visibility through initiatives such as joint research projects and scholar mobility programs.

Abstract:Background Lineage 7 PRRSV-2 is prevalent in some regions of China. However, the lack of specific differential diagnostic methods for this strain poses challenges for clinical prevention, control and epidemiological surveillance.Objective To establish an efficient and rapid reverse transcription real-time quantitative PCR (RT-qPCR) method for detecting lineage 7 PRRSV-2, thereby providing a reliable diagnostic tool for the early detection and monitoring of lineage 7 PRRSV-2 in swine herds.Methods A pair of specific primers and corresponding probes were designed based on the conserved sequence of the non-structural protein 2 (NSP2) gene of PRRSV-2. The final concentrations of primers and probes, as well as the annealing temperature, were then adjusted via the control variable method. Subsequently, the established method was evaluated in terms of the sensitivity, specificity, repeatability, and performance in detection of clinical samples.Results The developed RT-qPCR assay exhibited a good linear relationship within the range of 1×10¹ to 1×10⁹ copies/μL of the plasmid standard, with a correlation coefficient (R²) greater than 0.99. The minimum limit of detection of RT-qPCR method was 5 copies/μL. Specificity tests showed that this method only exhibited positive reactions to lineage 7 PRRSV-2, with no cross reaction with other lineages of PRRSV-2 and major swine disease pathogens, indicating high specificity. In the repeatability assays, the coefficients of variation (CV) for both intra-assay and inter-assay were all less than 1.00%, demonstrating the good stability of the established method. Furthermore, the detection results of clinical samples indicated that the RT-qPCR method had a higher positive detection rate than conventional PCR.Conclusion The RT-qPCR method established in this study is highly sensitive, specific, stable, accurate, and time-consuming. It is suitable for the rapid detection and quantitative analysis of lineage 7 PRRSV in clinical samples, providing technical support for the epidemiological surveillance and control of lineage 7 PRRSV-2.

Abstract:Background Contagious ecthyma is a zoonotic disease caused by infection with the Orf virus (ORFV). It is prevalent in sheep and goat flocks worldwide and seriously impacts the development of the sheep and goat farming industry. Objective To establish and evaluate a blocking enzyme-linked immunosorbent assay (ELISA) for detecting ORFV antibodies. Methods A monoclonal antibody (mAb) against the ORFV F1L protein was prepared and conjugated with horseradish peroxidase (HRP). With the F1L protein expressed in a baculovirus system as the detection antigen and the HRP-conjugated F1L mAb as the detection antibody, a blocking ELISA for ORFV antibody detection was established after optimization of the reaction conditions. The applicability of this method was assessed by testing 100 serum samples in parallel with an indirect immunofluorescence assay (IFA). Results The HRP-labeled F1L mAb exhibited a high titer and stable properties. The F1L protein was successfully expressed in the soluble form, with the purified F1L protein showing high purity and good reactivity. The optimal conditions for the blocking ELISA were as follows: coating concentration of F1L protein at 1.0 μg/mL and dilution of the HRP-conjugated antibody at 1:2 000. The cut-off values A and B were determined as 37.47% and 28.40%, respectively. Samples with an inhibition ratio greater than 37.47%, less than 28.40%, and between the two values were considered positive, negative, and doubtful, respectively. The intra-assay coefficients of variation (CV) did not exceed 4.49%, and the intra-assay CV did not exceed 6.28%, indicating good repeatability. The blocking ELISA showed the limit of detection of 1:128 for ORFV-positive serum, demonstrating higher sensitivity than indirect IFA. The method showed good specificity, with no cross-reactivity with antibodies against other common pathogens. Clinical sample testing revealed a 97.87% concordance rate between this method and indirect IFA. Conclusion A blocking ELISA based on the F1L protein is successfully established and can be used for the detection of ORFV antibodies.

Abstract:Background Microbial contamination control of drugs, foods, and cosmetics is central to ensuring quality and safety, while the plate count method is currently the most widely used approach for microbial limit testing. However, certain differences still exist between domestic and international standards for plate counting.Objective To clarify the differences in counting rules, error evaluation, rounding and reporting requirements among domestic and international standards for microbial plate counting in the fields of pharmaceuticals, food, and cosmetics, as well as their impacts on test results, so as to furnish scientific, evidence for the standardized implementation of microbial counting rules of the Chinese Pharmacopoeia (2025 Edition) and the improvement of the consistency and comparability of detection data across domestic laboratories.Methods Technical requirements of domestic and international standards were systematically sorted out. A multi-dilution series parallel plate counting model was established with Staphylococcus aureus as the test strain. The error control effects of different standards were evaluated based on indicators including the log-likelihood ratio statistic, inter-plate error rate, and relative standard deviation. The data dispersion performance was compared between the “rounding off” and “rounding up” rules, and the impact ranges of different reporting rules on counting results were verified.Results Significant differences were observed in the limit values for counting error judgment among different standards, resulting in the same plate data being judged as qualified under some standards and unqualified under others. The “rounding off” rule had slightly lower standard deviation (SD=0.34) than the “rounding up” rule (SD=0.41), demonstrating better data dispersion performance. In the scenario of multi-dilution counting, the maximum deviation caused by different reporting rules could reach 9.6%.Conclusion The differences in rules among domestic and international standards for microbial counting can lead to significant deviations in counting results, affecting the accuracy of test results and cross-standard comparability. On the basis of experimental data, it is recommended that statistical verification methods be introduced into the error judgment of microbial counting rules in Chinese pharmaceutical standards, “rounding off” be adopted as the rounding rule, and the weighted calculation logic of reporting rules be optimized. These measures are expected to achieve scientific alignment with international mainstream standards and enhance the consistency of quality supervision and the level of mutual recognition in international trade for related products in China.