Thermophilic cyanobacteria, as primary producers in extreme high-temperature ecosystems, has evolved unique adaptive mechanisms under high-temperature (> 45 ℃) and mineral-enriched extreme conditions in hot springs. For carbon assimilation, their dependence on ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) and its carboxysome microcellular compartment overcomes the inhibition of the enzyme activity at high temperatures by enhancing substrate affinity and CO₂ concentration efficiency. At the level of energy metabolism, the thermally stabilized photosystem Ⅱ (PSⅡ) complex and the electron transport chain are reconstructed to maintain the synergy between light and dark reactions at high temperatures. In terms of stability regulation, metabolic homeostasis is achieved through dynamic glycogen storage, antioxidant defense, and molecular chaperone networks. Systematic analysis of the carbon metabolism-environmental adaptation coupling mechanisms, carboxysome structural dynamics, and PSⅡ thermal protection mechanisms of thermophilic cyanobacteria will facilitate synthetic biology-driven engineering of thermophilic carbon-fixing chassis cells (e.g., heterologous expression of thermophilic Rubisco or design of thermostable electron transport chains), thereby expanding the direct bioconversion of high-temperature industrial waste gas and the biotreatment of heat-containing wastewater and promoting the innovation of micro biotechnology for extreme environments in pursuit of carbon neutrality.

As the research on microbiomes advances, the health hazards of endotoxins, components of the cell wall of Gram-negative bacteria, have garnered increasing attention. These toxins are released into the surrounding environment after bacterial death and lysis and pose a serious threat to human health through a variety of pathways. In response to this problem, probiotics, as non-pathogenic live microbial agents, have been widely used in the prevention and treatment of endotoxin-associated injuries in the past decades. As an extension and expansion of probiotic research, postbiotics, by virtue of their inactive organisms, have demonstrated significant advantages in terms of stability and safety, providing new solutions for the prevention and treatment of endotoxin-related diseases. This article introduces the sources, action pathways, and symptomatic manifestations of endotoxins, summarizes the definition, endotoxin-removing mechanisms, and application prospects of probiotics, and reviews the research progress in postbiotics regarding the endotoxin-removing mechanisms and the application in endotoxin removal via anti-inflammatory, intestinal flora-regulatory, and epithelial barrier-enhancing effects. It is expected to provide insights for the researchers and practitioners in the related fields.

Bacterial infections pose a serious threat to global public health, underscoring the urgent need for the development of novel antimicrobial agents. Antimicrobial peptides (AMPs), as natural products of the innate immune system, offer promising alternative therapies due to their broad-spectrum antibacterial activity, membrane-targeting mechanisms, and low propensity for inducing resistance. AMPs are derived from diverse sources, including animals, plants, and microorganisms. In recent years, with advances in technologies such as artificial intelligence, de novo design has also emerged as an important source of AMPs. The primary antibacterial mechanism of AMPs is believed to involve membrane disruption, with barrel-stave, carpet, and toroidal pore models proposed to explain this process. Beyond membrane disruption, AMPs can target bacterial cell walls, interact with intracellular targets, and modulate host immunity. In addition, AMPs exhibit antibiofilm activity through inhibition of quorum sensing, adhesion, and biofilm matrix synthesis of bacteria. This review provides a comprehensive overview of the sources and the antibacterial and antibiofilm mechanisms of AMPs, offering a reference for delving into the mechanisms of action of AMPs and guiding the rational design of novel AMPs based on the mechanisms.

Klebsiella pneumoniae, a major clinical pathogen, can cause various severe infectious diseases. In recent years, the emergence of multidrug-resistant hypervirulent K. pneumoniae strains has posed substantial challenges to clinical treatment and has become a serious threat to global public health. Depolymerases encoded by K. pneumoniae phages, through their unique mechanism of specifically degrading bacterial capsular polysaccharides, offer multiple advantages over conventional phage therapy. These advantages include evading host immune recognition, reducing the probability of horizontal gene transfer, and enhancing pharmacokinetic properties. Accordingly, bacteriophage depolymerases have now become a core direction for the development of innovative therapeutic strategies against multidrug-resistant bacterial infections. This article comprehensively summarizes the structural and functional characteristics of existing depolymerases, assesses their potential as alternative therapeutic strategies against multidrug-resistant K. pneumoniae infections, and explores the future application directions and modification strategies for K. pneumoniae phage depolymerases.

On the basis of microbial mineralization mechanisms, the in situ synthesis of nanomaterials by dissimilatory iron-reducing bacteria has demonstrated promising efficacy in enhanced oil recovery (EOR)—specifically microbial enhanced oil recovery (MEOR)—applications in recent years, and has gradually become a key focus of the research on MEOR. This paper, beginning with an analysis of the ecological diversity of dissimilatory iron-reducing bacteria within reservoirs and their adaptability to depositional environments, systematically reviews the nucleation mechanisms by which these organisms generate iron-oxide nanoparticles in situ and elucidates their roles in improving crude oil recovery. Dissimilatory iron-reducing bacteria produce iron-oxide nanoparticles via two distinct pathways: intracellularly controlled mineralization and extracellular induced mineralization. The former relies on enzyme-mediated reactions coupled with biomolecular template regulation, while the latter proceeds through extracellular electron transfer (EET) and coordination with extracellular polymeric substances (EPS) to precipitate metal ions. Studies indicate that nanoparticles biosynthesized by dissimilatory iron-reducing bacteria, owing to their inherently high specific surface area and wettability-reversal capabilities, are able to reduce oil-water interfacial tension and enhance emulsification, thereby improving oil displacement efficiency even in the absence of other microbial metabolic byproducts. Laboratory physical-model experiments have demonstrated that this biogenic nanoparticle-based EOR technique can boost recovery by 10%-15% in low-permeability and heterogeneous reservoir analogues. Compared with conventional approaches that rely on exogenous injection of pre-formed nanoparticles, the in situ biosynthesis route exhibits superior nanoparticle dispersion and interfacial modulation under experimental conditions, and offers the potential for sustained product generation in favorable reservoir niches. Nonetheless, the practical applicability and scalability of this strategy under true reservoir conditions remain to be fully validated through further field-scale studies and pilot tests.

The dynamic interactions between Staphylococcus and the host immune system determine the diversity of the pathogenic characteristics of Staphylococcus. During the invasive phase of infection, Staphylococcus breaches the host's innate immune barriers by releasing α-toxin, proteases, and superantigens, which compromise neutrophils, degrade complement proteins, and induce hyperactivation of immune responses. Following invasion and colonization, Staphylococcus establishes physical barriers through biofilm formation, evades immune recognition by generating small colony variants (SCVs), or infiltrates host cells to escape immune clearance. In acute infections, the pathogen exhibits aggressive traits via virulence factor secretion, whereas during chronic persistent infections, it adopts passive defense mechanisms through immune evasion. On the basis of these mechanisms, novel prevention and therapeutic strategies must simultaneously inhibit virulence factor production and block immune evasion pathways. Integrating our previous studies, this article systematically reviews the Staphylococcus-host immune system interactions and immune evasion mechanisms, aiming to provide insights for combating staphylococcal infections.

[Background] Prolyl oligopeptidases are serine proteases that cleave peptide bonds at the carboxyl side of proline. They have important applications in the food industry. However, the available prolyl oligopeptidases are limited, and their production levels are low. [Objective] To investigate the enzymatic properties of the peptidase family S9 prolyl oligopeptidase (MaPOP) from Microbulbifer arenaceous. [Methods] MaPOP was heterologously expressed in Escherichia coli. After purification by affinity chromatography, MaPOP was characterized and used for the preparation of ACE inhibitory peptides. [Results] MaPOP demonstrated the highest activity at pH 7.5 and 37 ℃. It was stable in the range of pH 3.5-11.0 and at temperatures up to 40 ℃. In the presence of 1.5 mol/L NaCl, MaPOP showed the highest enzyme activity, which was twice that in the absence of NaCl. The serine protease inhibitor PMSF and metal ions Co²⁺, Hg²⁺, and Zn²⁺ showed inhibitory effects on MaPOP. MaPOP specifically recognized proline residues in oligopeptides and cleaved the peptide bonds at the carboxyl side of internal L-prolines rather than those at the carboxyl side of D-prolines or between two proline residues. MaPOP was used in combination with flavor protease, neutral protease, AopepA, and AoproS8 to hydrolyze tilapia scales, achieving ACE inhibition rates of 55.85%-87.71%. [Conclusion] The heterologous expression and enzymatic characterization of MaPOP provide theoretical support for its application in the food industry.

[Background] Epimedium pubescens Maxim., a traditional medicinal plant, contains flavonoids as its primary bioactive constituents. The bioavailability of certain highly glycosylated flavonoids is limited due to high glycosylation. Biotransformation enables directional preparation of low-glycosylated flavonoids, representing a potential strategy to enhance the druggability of active components. [Objective] To screen functional strains capable of efficiently converting epimedin C to icariin, characterize their key enzyme systems, and improve substrate tolerance/conversion efficiency through microbial domestication. [Methods] We isolated strains from the rhizosphere microbiota and the phyllosphere microbiota of Epimedium by habitat-specific sampling. A thin layer chromatography (TLC)-HPLC system was established for the monitoring of transformation products. The phylogenetic analysis (16S rRNA gene) and glycosidase activity profiling were employed to identify the strain and functional enzymes, respectively. Single-factor experiments were conducted to optimize the crude enzyme reaction parameters (temperature, pH, substrate concentration, and enzyme activity). Six rounds of gradient stress domestication (0.01-0.12 g/mL aqueous extract of Epimedium) were implemented to evaluate the adaptive evolution of the strain. [Results] Bacillus sp. MY202402-3 was isolated, with its crude enzymes demonstrating notable α-L-rhamnosidase (18.7 U/mL) and β-glucosidase (3.3 U/mL) activities. In the medium containing 0.01 g/mL aqueous extract, the average epimedin C conversion rate and the average baohuoside Ⅰ yield reached 88.10% and 21.13%, respectively. The optimal reaction conditions of the crude enzyme were 50 ℃, pH 7.5, 1.2 mg/mL substrate, and 10 U/mL enzyme, under which the epimedin C conversion rate and the icariin purity reached 95.20% and 96.78%, respectively, within 2 h. Post-domestication, the conversion rate improved to 90.93% in the medium containing 0.10 g/mL aqueous extract, and the baohuoside Ⅰ yield reached 26.96% in the system containing 0.08 g/mL aqueous extract of Epimedium. [Conclusion] The integrated microbial screening-domestication strategy significantly enhances epimedin C biotransformation efficiency, providing insights for improving the bioavailability of herbal extracts. Concentration-dependent optimization demonstrates scalable industrial potential. As the Bacillus strain reported for Epimedium flavonoid transformation, MY202402-3 expands the application scope of Bacillus.

[Background] Biodenitrification of wastewater containing heavy metals under low temperature conditions is facing severe challenges. Copper ions (Cu²⁺) can significantly affect the denitrification efficiency of microorganisms. At present, the research remains limited regarding the denitrification characteristics and tolerance mechanism of psychrotolerant denitrifiers under Cu²⁺ stress. [Objective] To investigate the denitrification characteristics and Cu²⁺ tolerance of Comamonas aquatilis NY3, providing theoretical guidance for the biological treatment of low temperature wastewater containing Cu²⁺. [Methods] We conducted batch experiments to study the effects of different concentrations of Cu²⁺ on the nitrogen removal efficiency of strain NY3. The activities of the electron transfer system and the key enzymes in nitrogen metabolism during the transformation of inorganic nitrogen by strain NY3 were studied under high-concentration Cu²⁺. In addition, we determined whether exogenous sulphide addition helped to alleviate the inhibition on the growth and metabolism of this strain by Cu²⁺. [Results] Cu²⁺ significantly affected the nitrogen removal ability of strain NY3. When Cu²⁺ was not added, the removal rates of ammonium nitrogen, nitrate nitrogen, and nitrite nitrogen by strain NY3 were 99.98%, 100.00%, and 100.00%, respectively. After the addition of 8.0 mg/L Cu²⁺, the removal rates decreased to 51.56%, 55.70%, and 0, respectively. At the same time, the activity of the electron transfer system also decreased from 0.306, 0.115, and 0.124 μg/(g·min) to 0. The activities of ammonia monooxygenase and nitrate reductase decreased from 0.260 U/mg and 0.610 U/mg to 0.250 U/mg and 0.260 U/mg, respectively. The addition of 45.0 mg/L NaHS and 50.0 mg/L Na₂S significantly alleviated the inhibition of Cu²⁺ stress on the growth and metabolism of strain NY3. Specifically, 145 mg/L NaHS increased the removal rates of ammonium nitrogen and nitrate nitrogen by 48.17% and 70.94%, respectively. [Conclusion] Strain NY3 has certain tolerance to Cu²⁺ stress. Excessive Cu²⁺ has inhibitory effects on heterotrophic nitrification and aerobic denitrification of strain NY3. The inhibition of strain NY3 by Cu²⁺ can be alleviated by the addition of appropriate amounts of exogenous sulphur, which thereby increases the denitrification efficiency under Cu²⁺ stress. The findings provide a theoretical basis for the treatment of nitrogen-contaminated wastewater containing Cu²⁺ by microorganisms in a low-temperature environment.

[Background] Rhizosphere microorganisms are commonly employed in soil heavy metal remediation due to their ability to enhance soil fertility and boost crop growth. However, the efficacy of a solitary microbial agent in remediation is inconsistent and vulnerable to interference from indigenous microorganisms. Consequently, developing reliable composite microbial agents is crucial for facilitating plant remediation of heavy metals. [Objective] We conducted pot experiments to the study mechanism by which Burkholderia sp. DHC34 and/or Pseudomonas fluorescens fortified Ageratum conyzoides L. in the remediation of cadmium (Cd)-contaminated soil. [Methods] The single microbial agents BH (Burkholderia sp. DHC34) and PH (P. fluorescens), as well as the composite microbial agents BPH1-1, BPH1-2, and BPH2-1 (consisting of Burkholderia sp. DHC34 and P. fluorescens mixed at ratios of 1:1, 1:2, and 2:1, respectively), were used to treat A. conyzoides. The plants treated with sterile water were taken as the control group. The plant biomass and Cd accumulation were measured, and the micro-method was employed to determine the activities of antioxidant enzymes in plant leaves. Microbial diversity was assessed through Illumina PE250 high-throughput sequencing. [Results] In comparison to the control group, P. fluorescens alone significantly enhanced the activities of peroxidase, catalase, and superoxide dismutase, while increasing the plant height and fresh weight by 50.0% and 77.7%, respectively. Burkholderia sp. DHC34 resulted in weak acid extractable fraction of Cd comprising 60.0% of the total, while increasing the Cd accumulation per A. conyzoides plant by 188% relative to the control. The co-inoculation of the two strains in varying ratios significantly enhanced the Cd uptake by A. conyzoides plants. BPH1-2 exhibited the most significant performance, increasing the biomass and Cd accumulation of A. conyzoides by 71.2% and 166%, respectively. The bioenrichment coefficients of aboveground and underground parts reached 11.40 and 6.25, respectively. Furthermore, the co-inoculation of both strains induced substantial changes in the rhizosphere microbial community composition, markedly increasing the abundance of beneficial taxa such as Flavisolibacter, Adhaeribacter, and Streptomyces. By regulating the activity of the microbial groups involved in carbon and nitrogen cycling, the composite microbial agents fortified the Cd remediation efficiency and stablized the rhizosphere environment of A. conyzoides. [Conclusion] Burkholderia sp. DHC34 and P. fluorescens exhibit potential applications as growth-promoting agents for A. conyzoides. The co-inoculation of the two bacterial strains could create a functional pathway synergizing the plant growth-promoting effect of P. fluorescens and the Cd activing effect of Burkholderia sp. DHC34. The co-inoculation leads to the enrichment of beneficial rhizosphere microbial groups to mitigate Cd stress on A. conyzoides. Notably, the overall efficacy of BH surpassed that of PH, and BPH1-2 exhibited the best performance among the composite agents.

[Background] In the context of globalization and rapid advancements of biotechnology, the biosafety risks associated with microorganisms have aroused increasing concern, integrated into the national security system. Urumqi is a key node city in northwest China, and its wastewater treatment plants contain biosafety markers such as pathogenic microorganisms, which can serve as early warning signals for potential threats. [Objective] This study focuses on the microbial communities in the wastewater treatment plants of Urumqi. By analyzing the structural characteristics, diversity distribution, and potential biological risks of these communities, we aim to provide a scientific foundation for preventing the transmission of cross-border pathogens and maintaining regional bio-security. [Methods] Water samples were collected at a depth of 0.5 m from each of nine typical wastewater treatment plants in Urumqi in August 2 024. After processing, the samples underwent metagenomic sequencing. R (v4.4.2) was used for statistical analyses. Specific R packages were employed to calculate the α-diversity indices of bacteria and viruses, and non-parametric Kruskal-Wallis H tests were conducted to identify differences among the samples. Principal coordinate analysis (PCoA) based on Bray-Curtis distance and ANOSIM tests were performed to further understand the variations in microbial community structures across different sample groups. [Results] At the genus level, 300 microbial genera were detected, including 282 bacterial genera (94%), 2 fungal genera (0.67%), and 16 viral genera (5.33%), among which Aliarcobacter and Bacteroides were dominant. At the species level, 864 microbial species were identified, among which Aliarcobacter cryaerophilus and Cetobacterium somerae had high relative abundance. Alpha diversity analysis showed that the wastewater treatment plants S5 and S7 with integrated treatment processes had higher microbial diversity (Shannon indices of 3.398 and 3.421, respectively). Beta diversity analysis indicated overall similar microbial community structures across three sample groups, with no significant differences (ANOSIM R²=0.32, P > 0.05), though sample S4 deviated. Cluster heat-map analysis confirmed spatial distribution differences in microbial survival adaptability at various sampling points, with S9 and S1 having more positively correlated genera and different numbers of such genera at each point. The unique combination of arid hypersalinity, large low-temperature fluctuations, and a high-protein diet drove marked enrichment of halotolerant Arcobacter and psychrophiles. [Conclusion] The microbial communities in the nine wastewater treatment plants in Urumqi are dominated by bacteria, with specific genera and species. The plants with integrated treatment demonstrate higher microbial diversity and more pronounced spatial distribution differences in community structure. These findings provide valuable baseline data for regional biosurveillance and assessment of urban ecosystem health.

[Background] Pymetrozine is a pesticide that exhibits stability under alkaline conditions and possesses potential carcinogenic properties. Most existing strains capable of degrading pymetrozine have been isolated from highly contaminated environments or pesticide production facilities. [Objective] To obtain strains suitable for bioremediation of pymetrozine-contaminated alkaline soil. [Methods] We collected soil samples from long-term continuous cropping cotton fields in Xinjiang. Pymetrozine-degrading bacteria were isolated by enrichment followed by purification. Subsequently, their degradation characteristics, metabolic pathways, and key genes were investigated. [Results] An efficient pymetrozine-degrading bacterial strain 3BR11-2 was successfully isolated. This strain could completely degrade pymetrozine when it was inoculated in a 250 mL Erlenmeyer flask containing 100 mL basic inorganic salt medium (50 mg/L pymetrozine, pH 8.0) at an inoculum size of 2% (OD₆₀₀=1.0) and cultured at 37 ℃ and 120 r/min for 36 h. UHPLC-Q-TOF/MS revealed the production of three metabolites during the degradation process by strain 3BR11-2. These metabolites were identified as 3-pyridinemethanol, nicotinic acid (NA), and 6-hydroxynicotinic acid (6HNA). The phylogenetic analysis based on 16S rRNA gene sequence comparison showed that the strain shared 99.29% similarity with Nocardioides simplex. With the genome of strain 3BR11-2 as a template, primers were designed based on the pymetrozine hydrolase gene pyzH from strain Pseudomonas sp. BYT-1. The gene pyzH was then amplified by PCR, which yielded a fragment of 846 bp. This fragment shared the similarity of 99.65% (843/846 nucleotides) with pyzH. Furthermore, amino acid sequence alignment indicated that the pymetrozine hydrolase expressed by pyzH of strain 3BR11-2 had three residue changes: A195S, C212S, and A263T. [Conclusion] This study enriches the repertoire of highly efficient pymetrozine-degrading bacteria and holds promise for the application of bioremediation strategies targeting pymetrozine in unique saline-alkali environments.

[Background] Saline-alkali land is an important land resource in China. Using microorganisms to improve soil environment and promote plant growth has become a research focus in recent years. [Objective] A strain of saline-tolerant growth-promoting bacteria was selected from rice root soil in the saline-alkali land in Daqing, Heilongjiang. This study is expected to provide elite strain resources for the development of microbial agents suitable for rice growth in the saline-alkali land of Northeast China. [Methods] Saline-tolerant microbial strains were screened by microbial isolation and culture. These strains were subjected to colony morphology observation, physiological and biochemical tests, growth curve determination, and 16S rRNA gene identification. The multifunctional identification media were used to evaluate the phosphorus solubilization, siderophore production, amylase production, and salt-alkali tolerance of the target strain. The disease resistance of the strain was tested by the plate confrontation method. The abilities of the strain to produce extracellular polymetric substance (EPS) and indole-3-acetic acid (IAA) and reduce alkali were examined. Furthermore, the rice growth-promoting effect of the strain was evaluated by rice seed germination and water culture experiments. [Results] A saline-alkali tolerant strain J-009 was screened from the rice root soil in the saline-alkali land in Daqing. The strain was rod-shaped and Gram-positive, with white and wrinkled colonies. The strain reached its growth peak 12 h after inoculation, survived within the range of pH 8.0–10.0, and showed tolerance to the maximum salt (NaCl) concentration of 5%. Strain J-009 was capable of solubilizing inorganic phosphorus and organophosphorus and producing siderophores, amylase, and H₂S. It can utilize glucose, arabinose, and sorbitol but cannot utilize sucrose or maltose. The reactions of peroxidase, urease, and cellulase were all positive. Strain J-009 was identified by 16S rRNA gene sequence analysis as Bacillus halotolerans, with the GenBank accession number PV635949. Strain J-009 had a certain inhibitory effect on rice blast. This strain showed the EPS production of (0.581±0.083) g/g, the IAA secretion of (12.39±0.66) mg/L, and the alkali-reducing capacity of 13.9%. Compared with the control group, the strain J-009 treatment significantly increased the root length and plant height of rice seedlings. [Conclusion] Strain J-009 demonstrated good saline-alkali tolerance and plant growth-promoting effect, and it could effectively promote the growth of rice in saline-alkali environment. The findings lay a foundation for the microbial improvement of saline-alkali soil and the development of microbial agents in the future.

[Background] In 1984, two novel halophilic archaea species, Halobacterium dachaidanensis sp. nov. and Hbt. tangguensis sp. nov., were isolated from an inland saline lake and a marine solar saltern in China, and first published on Acta Microbiologica Sinica by Chinese researchers. Over the past four decades, the taxonomic system of halophilic archaea has been continuously updated, while the precise taxonomic status of these two species remains unclear. [Objective] To clarify the taxonomic status of Hbt. dachaidanensis F3 and Hbt. tangguensis F5 by phylogenomic and comparative genomic analyses and phenotypic characterization. [Methods] We performed the phylogenetic analysis based on amplified and sequenced 16S rRNA and rpoB' genes. The draft genomes of these two strains were sequenced and the taxonomic status of them was elucidated by phylogenomic and comparative genomic analyses. The major metabolic pathways of both strains were identified through KEGG annotation. The key physiological and biochemical characteristics were comparatively analyzed via phenotypic assays. [Results] Phylogenetic analyses revealed that strains F3 and F5 formed tight clusters with Har. amylolytica BD-3^T and Natrinema thermotolerans JCM 11050^T, respectively. The genomic G+C content of strains F3 and F5 was 62.2% and 65.5%, respectively. Comparative genomic analysis showed that the average nucleotide identity (ANI), digital DNA-DNA hybridization (dDDH), and average amino acid identity (AAI) between strain F3 and Har. amylolytica BD-3^T were 98.3%, 88.1%, and 97.9%, respectively, and these three values between strain F5 and Nnm. thermotolerans JCM 11050^T were 98.0%, 85.2%, and 97.9%, respectively. These values were higher than the thresholds for species demarcation in prokaryotes. The phenotypic characterization showed that the optimum growth salinity, temperature, and pH for strain F3 were 2.6 mol/L, 37 ℃, and 7.5, while those for strain F5 were 3.4 mol/L, 40 ℃, and 7.0, respectively. Diverse differential phenotypic characteristics were found among these two strains and their related species. Functional gene annotations identified carbohydrate metabolism and amino acid metabolism as the predominant metabolic pathways in both strains. [Conclusion] The taxonomic status of Hbt. dachaidanensis F3 and Hbt. tangguensis F5 was reclarified by polyphasic taxonomic characterization as Har. amylolytica and Nnm. thermotolerans, respectively. Strain F3 may represent a novel subspecies, for which the name, Haloarcula amylolytica subsp. dachaidanensis subsp. nov., is proposed.

[Background] The transcription factor ACE1 is widely present in cellulase- producing filamentous fungi, while its regulatory function in Aspergillus niger remains unclear. [Objective] To investigate the regulatory effects of ACE1 on β-glucosidase (BGL) expression, mycelial development, and pigment biosynthesis by deleting and overexpressing ACE1 in A. niger. [Methods] The knockout strain Δace1 and overexpression strain Oace1 were constructed with the parent strain An-1 through CRISPR-Cas9 gene editing. The BGL activity, mycelial growth, sporulation, and yellow pigment were determined under various induction conditions. [Results] The phylogenetic analysis revealed low homology of ace1 between A. niger and Trichoderma reesei or Neurospora crassa. The mutant Δace1 was successfully constructed after introduction of the CRISPR-Cas9 plasmid and the donor fragment. When microcrystalline cellulose (MCC) and corncob powder were used as inducers, the BGL activity of Δace1 with cellobiose as the substrate decreased by 86.1% and 54.9%, and that of Δace1 with p-nitrophenyl-α-D-glucopyranoside (pNPG) as the substrate decreased by 23.7% and 63.98%, respectively. The overexpression cassette P_gpd-ace1-T_gla was precisely integrated into the kusA locus to construct Oace1. The BGL activity of Oace1 was 1.37-2.16 times that of the wild strain An-1 with MCC as the inducer. On the medium with stevioside or MCC as the carbon source, Δace1 showed delayed hyphal extension and conidiophore differentiation, while Oace1 retained normal morphology. The yellow pigment biosynthesis ability of Δace1 was significantly enhanced in the solid and liquid media containing stevioside. [Conclusion] The transcription factor ACE1 positively regulates BGL expression and sporulation, while it negatively regulates yellow pigment synthesis in A. niger. Strengthening the expression of ACE1 effectively improves BGL production. This study provides a new strategy for optimizing the enzyme production performance of A. niger.

[Background] Cyperus esculentus, a high-yield cash crop with remarkable stress and barren soil tolerance and broad adaptability, serves multiple purposes including grain production, oil extraction, forage production, medicinal applications, and ecological landscaping. Its cultivation holds significant potential for rehabilitating saline-alkali abandoned farmlands in Xinjiang. [Objective] To isolate salt-tolerant plant growth-promoting rhizobacteria (PGPR) with high indole-3-acetic acid (indole-3-acetic acid, IAA) production from the saline-alkali sandy soils in Xinjiang to enhance C. esculentus tolerance to moderate-to-high salt stress, thereby providing microbial resources for soil amelioration. [Methods] Bacteria were screened via selective culture, shake-flask fermentation, and 16S rRNA gene sequencing. The plant growth-promoting effects of these bacteria were determined. The plant growth-promoting effects of these bacteria were then examined through seed germination and pot experiments with C. esculentus under salt stress. [Results] Four IAA-producing strains were isolated as Y8, Y35, Y46, and Y4-20, which were identified as Bacillus rugosus, Bacillus aryabhattai, Bacillus halotolerans, and Halomonas alkaliantarctica, respectively. All the four strains demonstrated salt tolerance, nitrogen fixation, and inorganic phosphate solubilization. Furthermore, Y35, Y46, and Y4-20 exhibited potassium-solubilizing ability. The results from both seed germination and pot experiments demonstrated that all the four IAA-producing salt-tolerant PGPR strains significantly promoted the seed germination and seedling growth of C. esculentus under salt stress. Notably, strain Y8 exhibited particularly outstanding promotion effects. [Conclusion] The saline-alkali soils in Xinjiang harbor salt-tolerant PGPR with high IAA production, which can significantly enhance the salt tolerance of C. esculentus under moderate-to-high salt stress. This study provides valuable microbial resources for developing bio-fertilizers to ameliorate saline-alkali soils in Xinjiang.

[Background] Gut bacteria play crucial roles in the development, nutrient metabolism, and immune defenses of host insects, with their community composition being significantly influenced by diet sources. However, the research on the diversity of gut microbiota in Lasioderma serricorne larvae under different dietary conditions remains limited. [Objective] To elucidate the intrinsic relationship between gut bacterial diversity and diet sources in L. serricorne larvae and isolate and identify culturable symbiotic bacteria. [Methods] Full-length 16S rRNA gene sequencing was performed on the PacBio SMRT platform to analyze the community structure of gut bacteria in L. serricorne larvae in the artificial feed (SL) group, tobacco domestication (YC) group, and wild environment (WF) group. RT-qPCR was employed to quantify the expression levels of three symbiotic bacteria, Enterobacter, Wolbachia, and Arsenophonus, under the three dietary conditions. Culturable dominant bacteria were isolated and identified via four functionally distinct media. [Results] The number of gut microbiota OTUs varied significantly among different groups (SL: 174 OTUs; YC: 115 OTUs; WF: 62 OTUs). Alpha diversity indices confirmed significant intergroup differences (SL > YC > WF, P < 0.05). At the genus level, the dominant bacteria were Enterobacter (61.43%), Wolbachia (13.12%), and Enterococcus (19.34%) in the SL group, Wolbachia (87.09%) and Enterococcus (11.88%) in the YC group, and Arsenophonus (97.91%) in the WF group. RT-qPCR validated the differential expression of Enterobacter, Wolbachia, and Arsenophonus across groups. Thirteen culturable strains were isolated and phylogenetically identified as Pantoea (SL1 and YC5), Acinetobacter (SL6, YC1, WF2, WF8, and WF10), Enterobacter (CB2), Exiguobacterium (SL5 and YC3), and Enterococcus (WF6, YCL, and WFL). [Conclusion] This multi-dimensional analysis demonstrates how diets shape the gut microbiota structure of L. serricorne larvae, providing new insights for developing gut microbiota-based eco-friendly strategies for pest management.

[Background] Fusarium oxysporum is a dominant pathogen for the root rot of ginseng, which has a wide spectrum of hosts and strong pathogenicity, causing devastating harm to cultivated ginseng in farmland. FoeIF1a of F. oxysporum is the key factor in the initiation of eukaryotic translation, and its research is of great significance for understanding the pathogenic mechanism of this fungus. [Objective] To explore the influences of FoeIF1a on the mycelial growth, spore yield, hyperosmotic and stress responses, toxin content, and pathogenicity of F. oxysporum f. sp. ginseng 0083 and clarify the gene functions. [Methods] We employed bioinformatics tools to predict the properties of FoeIF1a, and then constructed the knockout and complementary vectors of this gene. After the vectors were introduced by Agrobacterium tumefaciens-mediated transformation, the transformants were screened by molecular biology verification. We then analyzed the results by observing the phenotypes of the wild-type strain Fo0083, knockout strain, and complementary strain. [Results] The full-length FoeIF1a was 408 bp, encoding 135 amino acid residues. The growth rate, biomass, spore yield, and spore germination rate of ΔFoeIF1a were significantly lower than those of the wild-type strain Fo0083. ΔFoeIF1a was sensitive to pH 5.0, pH 7.0, pH 9.0, 800 mg/mL CR, 0.01% SDS, different carbon sources (1 mol/L glucose and 1 mol/L sucrose), oxidative stress (5 mmol/L H₂O₂), osmotic stress (1 mol/L NaCl, 1 mol/L KCl, and 1 mol/L sorbitol), and acid-base indicator (0.15% bromophenol blue). In addition, ΔFoeIF1a showed significantly increased content of fusarium acid and attenuated pathogenicity. The complementary strain ΔFoeIF1a-C approached the wild-type strain in the indicators above. [Conclusion] FoeIF1a in F. oxysporum can reduce the mycelium growth rate, biomass, spore yield, and spore germination rate, and it is sensitive to hyperosmotic stress, affecting the fusarium acid content and pathogenicity.

[Background] Dryland tomato is one of the characteristic industries in Huguan County, Changzhi City, Shanxi Province. In recent years, increased rainfall has led to frequent outbreaks of tomato early blight. [Objective] To identify the pathogenic fungi causing tomato early blight in the experimental field and analyze their biological characteristics, we introduced the adjuvant Jijian and screened the optimal dosage reduction proportion that minimize the usage of difenoconazole+azoxystrobin SC while maintaining the efficacy through indoor antifungal tests and field trials. [Methods] Morphological observation and molecular biological methods were employed to identify the isolated pathogen strain W1. The biological indicators including optimal carbon source, temperature, light conditions, and pH value of this strain were studied. The organic adjuvant Jijian was introduced, and indoor antifungal tests were conducted via the plate confrontation method to optimize the dosage reduction proportion of the fungicide. Furthermore, field trials were carried out to evaluate the control efficacy. [Results] Strain W1 was identified as Alternaria tenuissima. The optimal carbon source, temperature, and pH for the vegetative growth of this strain was fructose, 28 ℃, and 7.0, respectively, and the mycelial growth was fastest under complete darkness conditions. Indoor antifungal tests showed that all dosage reduction treatments of difenoconazole+azoxystrobin SC combined with Jijian had inhibitory effects on the fungus causing tomato early blight. The 10% reduction+Jijian treatment was superior to conventional dosage treatment, with an inhibition rate of 93.90%. In field trials, the control efficacy of the 10% reduction+Jijian and 20% reduction+Jijian treatments was 77.54% and 72.94%, respectively, 7 days after the first application and 83.40% and 80.32%, respectively, 7 days after the second application, surpassing that of the conventional application. [Conclusion] A. tenuissima is the pathogenic fungus causing tomato early blight in this experimental field. Adding the adjuvant Jijian can reduce the usage of difenoconazole+ azoxystrobin SC while maintaining the control efficacy against tomato early blight, with 20% reduction in difenoconazole+azoxystrobin SC+Jijian being the most suitable treatment.

[Background] Exploring salt-tolerant rhizosphere microorganisms is of great significance for improving and utilizing saline-alkali land and for alleviating plant salt stress. [Objective] To screen plant growth-promoting rhizobacteria (PGPR) of Leymus chinensis planted in saline-alkali soils, analyze their growth characteristics and plant growth-promoting effects, and explore their effects on seed germination of L. chinensis, thus providing a scientific basis for the development and application of PGPR. [Methods] The strains were isolated from the rhizosphere soil of L. chinensis. The salt tolerance and pH adaptability of the strains were tested in LB media with different salt concentrations and pH values. The ability to produce indole-3-acetic acid (IAA) was determined by the Salkowski colorimetric method. The molybdenum-antimony colorimetric method and CAS medium were used to assess the abilities of the strains to solubilize phosphorus and produce siderophores, respectively. The strains were identified based on morphological, physiological, biochemical, and 16S rRNA gene sequencing, and the dominant strains with plant growth-promoting effects were screened. Seed germination tests were conducted to verify the plant growth-promoting effects of the strains. Pot experiments on the interactions between strains and L. chinensis seedlings under salt stress were carried out to determine growth and physiological indicators of the seedlings under different treatments. [Results] Three efficient multifunctional strains with plant growth-promoting effects were screened out: Rossellomorea arthrocnemi Z7, Rossellomorea aquimaris Z25, and Cytobacillus oceanisediminis Z49. They could survive in LB media with salt concentrations of 0%–9% and pH 8.85–10. Z7, Z25, and Z49 showed the IAA production capacity of 10.10, 7.74, and 17.44 mg/L and the inorganic phosphorus-solubilizing capacity of 107.80, 128.52, and 95.95 mg/L, respectively. Among them, strain Z25 produced siderophores. Compared with the control group, Z7, Z25, and Z49 increased the seed germination rate by 5.45%, 16.36%, and 13.64% and the root length by 17.86%, 31.79%, and 27.14% respectively. Inoculation of the three strains reduced the MDA content and increased the content of antioxidant enzymes of L. chinensis seedlings in Pot experiments. [Conclusion] Strains Z7, Z25, and Z49 are multifunctional bacteria that produce IAA, solubilize phosphorus, and tolerate saline-alkali. They can serve as excellent strain resources for the development of bioinoculants and improve the utilization rate of saline-alkali soil.

[Background] Ralstonia solanacearum-induced tomato bacterial wilt is a major soil-borne disease in tomato cultivation, and effective control strategies remain limited. Screening antagonistic strains and developing biocontrol agents have become primary objectives in combating this disease. [Objective] To elucidate the growth-promoting effects, disease-preventing efficacy, and biocontrol mechanisms of Pseudomonas aeruginosa strain B-6 through laboratory and field trials and whole genome sequencing. [Methods] We employed the root irrigation method to evaluate the control efficacy of strain B-6 fermentation broth against tomato bacterial wilt, and then employed the seed soaking treatment to assess the growth-promoting effects of the strain on tomato seedlings. The second- and third-generation sequencing technologies were integrated to reveal the whole genome sequence of strain B-6. Gene functional annotation was performed via NR, Swiss-Prot, KEGG, COG, and GO databases. [Results] The results of laboratory and field experiments indicated that the control efficacy of the fermentation broth of strain B-6 decreased significantly as the dilution ratio increased. The 50-fold dilution group exhibited over 60% control effect against tomato bacterial wilt and could significantly promote the elongation of plumules and radicles. Genome sequencing showed that its total length was 6 456 367 bp, with the G+C content of 66.37%, containing 75 tRNA genes and 12 rRNA genes. Through combined annotation using multiple databases, 5 994 genes were successfully annotated. Among the 16 secondary metabolite synthesis gene clusters, 8 were completely matched with the synthesis gene clusters of known bacteriostatic compounds such as pyoluteorin, l-2-amino-4-methoxy-trans-3-butenoic acid, and pyocyanine, which suggested the molecular basis for its bacteriostatic activity. The coding region coding sequence (CDS) was predicted to be annotated to 5 865 genes, with 247 carbohydrate-active enzymes (CAZyme). [Conclusion] Strain B-6 demonstrates promising biocontrol activity against tomato bacterial wilt and promotes tomato growth. Its genome contains diverse antimicrobial metabolite biosynthetic gene clusters, which underscores the development potential of this strain as a high-efficacy biocontrol agent.

[Background] With the expansion of sorghum planting area in Shanxi Province, the occurrence of leaf spot becomes common, with aggravated harm and increasingly complex pathogen species. [Objective] To clarify the species and biological characteristics of the pathogen causing Pestalotiopsis leaf spot in sorghum in Shanxi Province, screen out effective fungicides that can effectively prevent and control the disease, and provide a theoretical basis for the identification and prevention of this disease in sorghum. [Methods] We employed the tissue culture method to isolate the pathogen and then confirmed the pathogen according to Koch's postulates. We identified the pathogen by combining morphological characteristics and molecular biological evidence, and adopted the mycelial growth method and dilution plate method to determine the biological characteristics and fungicide sensitivity of the pathogen. [Results] The morphological characteristics of the pathogen were consistent with those of Pestalotiopsis kenyana, and the ITS, TEF1-α, and TUB gene sequences of the pathogen showed the similarity above 99% compared with those of the P. kenyana standard strain CBS442.67. The phylogenetic tree constructed based on the ITS-TEF1-α-TUB gene sequences showed that the strain was in the same branch with P. kenyana. The suitable culture conditions for mycelial growth of the pathogen were 20-25 ℃, 24 h dark, pH 5.0-7.0, glucose as the carbon source, and peptone as the nitrogen source. Among the eight fungicides tested, 98% fludioxonil (0.105 4 mg/L), 98% pyraclostrobin (0.121 4 mg/L), 98% azoxystrobin (0.174 1 mg/L), 98% epoxiconazole (0.419 4 mg/L), and 98% tebuconazole (0.924 9 mg/L) showed stronger virulence on the mycelial growth of the pathogen strain L-GLYK1, with EC₅₀ values all less than 1.0 mg/L. Among them, 98% pyraclostrobin showed the best inhibitory effect on spore germination of strain L-GLYK1, with an EC₅₀ value of 0.010 6 mg/L. In addition, 98% tebuconazole (8.617 5 mg/L) and 98% epoxiconazole (7.527 2 mg/L) also exhibited good inhibitory effects. [Conclusion] P. kenyana is the pathogen causing Pestalotiopsis leaf spot in sorghum in Shanxi Province. The optimal culture conditions of this pathogen are determined, and 98% pyraclostrobin, 98% tebuconazole, and 98% epoxiconazole can be used for further experimental research on the prevention and control of this disease.

[Background] Sorghum serves as a crucial raw material for China's brewing industry. However, during harvest and storage, it is susceptible to infections by Alternaria, Fusarium, Curvularia, and Aspergillus, which lead to mold growth and mycotoxin contamination. These issues severely compromise the quality of brewing products and pose food safety risks. While chemical control methods may cause environmental pollution, biocontrol has emerged as a research hotspot due to its eco-friendly characteristics. [Objective] To screen the microbial strains exhibiting antagonistic effects against fungi infecting brewing sorghum, providing candidate strains for biocontrol agent development. Additionally, it sought to establish a theoretical foundation for improving China's brewing sorghum storage and fungal control systems. [Methods] Taking Alternaria destruens GN3, Fusarium equiseti GN5, Aspergillus chevalieri GN11, Curvularia pseudointermedia GN19, and Candida parapsilosis GN10 as indicator strains and using the plate confrontation method, we isolated the antagonistic strain MY1 and further identified this strain based on its morphological features, physiological and biochemical properties, and molecular evidence. The inhibitory activity of the sterile fermentation filtrate of this strain was assessed via the agar well diffusion assay, while that of volatile metabolites was evaluated via the double-plate confrontation method. Additionally, extracellular hydrolase production was detected. [Results] Strain MY1 exhibited significant inhibitory effects on all the five tested fungal strains, with the inhibition rates exceeding 50.00%. It was identified as Bacillus velezensis. The sterile fermentation filtrate inhibited the growth of A. destruens GN3 at a low concentration of 0.8%, causing abnormal mycelial morphology and achieving an inhibition rate of 53.95%. Furthermore, MY1 demonstrated protease and cellulase secretion capabilities and produced volatile antifungal compounds, displaying broad-spectrum inhibitory activity against multiple pathogenic fungi. [Conclusion] The antagonistic strain MY1 shows remarkable inhibitory activity against fungi infecting brewing sorghum and holds potential as a biocontrol agent for fungal contamination management.

[Background] Transglutaminases (TGases) play a significant role in the food industry, while their heterologous expression levels remain low. [Objective] This study aimed to achieve high-level expression of SmTGase from Streptomyces mobaraensis in Komagataella phaffii through a combinatorial strategy. [Methods] The combinatorial strategy involving co-expression of the propeptide and mature domains, co-expression of molecular chaperones, and overexpression of translation initiation factors was employed to enhance the expression level of SmTGase in K. phaffii. High-cell-density fermentation was carried out in a 5 L bioreactor with a glycerol-methanol co-feeding strategy for the efficient production of SmTGase. SmTGase was purified via a one-step strong anion-exchange column, and the enzymatic properties of SmTGase were characterized. [Results] The recombinant strain yielded an enzyme activity of 5.67 U/mL in a shake flask. Through the high-cell density fermentation in a 5 L bioreactor, the recombinant strain produced an enzyme activity of up to 80.5 U/mL with a protein titer of 7.68 g/L. The purified SmTGase exhibited the highest activity at pH 7.0 and 55 ℃, and it was stable within pH 5.5-8.0 and at temperatures below 45 ℃. [Conclusion] This study provides a valuable reference for the high-level expression of TGases in K. phaffii.

[Background] In the fermentation of the upper-layer fermented grains of Nongxiangxing Baijiu, there are widespread issues such as low raw material utilization rate and poor flavor quality. [Objective] To improve the yield and quality of Nongxiangxing Baijiu from the upper-layer fermented grains. [Methods] In this study, functional microbiomes (with high yields of amylase and ester compounds) were used for enhanced fermentation. [Results] The results showed that the enhancement with the functional microbiome increased the starch utilization rate in the upper-layer fermented grains by 23.6%, the content of ethanol in the crude Baijiu by 33.3%, and the content of ester flavor substances by 31.4%. At the same time, the functional microbiome significantly increased the relative abundance of Lactobacillus, Limosilactobacillus, Pichia, and Thermoascus while decreasing the relative abundance of Bacillus, Kroppenstedtia, and Aspergillus in the fermented grains. The changes in the relative abundance of these microbial genera led to significant differences in the content of 13 volatile compounds such as caproic acid, caprylic acid, ethyl caproate, and ethyl acetate in crude Baijiu. Spearman correlation analysis indicated that moisture, reducing sugar content, and acidity had significant positive correlations with the main microbial genera and were important driving factors in the fermentation process of fermented grains. Analysis indicated that the functional enzymes and their abundance of the microbial community in the fermented grains showed significant differences at different fermentation stages. It suggested that the microbial community changes in the fermented grains were the main cause of metabolic differences at different fermentation stages. [Conclusion] This study systematically investigated the effects of functional microbiome disturbance on the physical and chemical properties of the upper-layer fermented grains of Nongxiangxing Baijiu, the succession of the microbial community, and the synthesis of volatile flavor substances during the fermentation process, providing a reference for optimizing the brewing process of Nongxiangxing Baijiu through enhancement with functional microbiomes.

[Background] Pasteurella multocida is the major pathogen causing bovine respiratory diseases in China, and strains with multidrug resistance keep emerging. [Objective] To investigate the prevalence and drug resistance of bovine respiratory tract-derived P. multocida strains from different regions and evaluate the attenuating effects of traditional Chinese medicine (TCM) on the resistance of drug-resistant strains. [Methods] The pathogenic bacteria were isolated from the lung tissue samples and nasal swabs of diseased cattle and identified via biochemical and molecular methods. The K-B method was adopted to test the antimicrobial susceptibility, and resistance genes were identified by PCR. Subsequently, the test for attenuating effects on drug resistance was conducted. The minimum inhibitory concentrations (MICs) of Huhuang decoction and its components against the isolates were determined by the microdilution method. Strains were exposed to antimicrobials at 1/2, 1/4, and 1/8 MIC for 24, 48, and 72 h, and changes in their antimicrobial susceptibility were assessed. [Results] Ten suspected P. multocida strains were isolated, showing smooth, convex, and gray-white colonies on blood agar, with no hemolysis. Wright's staining showed that the cells were rod, with both ends stained. The biochemical test results of the strains were consistent with those of P. multocida, and the amplification product of kmt1 showed the expected length. Capsular serotyping identified 8 strains as type A and 2 as type D. Antimicrobial susceptibility test results revealed high resistance rates to streptomycin (70%), gentamicin (60%), ofloxacin (60%), and amoxicillin (50%). The resistance rates to amikacin, kanamycin, ciprofloxacin, norfloxacin, enrofloxacin, and trimethoprim-sulfamethoxazole ranged from 10% to 40%. Five strains showcased multi-drug resistance, accounting for 50% of the total strains. The resistance genes strA, strB, and bla_ROB-1 were identified in all the isolates, while aac(6')-Ib-cr, Sul1, tetB, and tetH were found in 60%, 50%, 30%, and 10% of strains, respectively. Huhuang decoction and Scutellariae Radix decoction attenuated the resistance to amikacin, kanamycin, ciprofloxacin, and enrofloxacin in a concentration- and time-dependent manner. The most pronounced attenutation of resistance was observed after 72 h-exposure to the decoctions at 1/2 MIC. [Conclusion] A total of 10 P. multocida strains were isolated, among which 50% of the strains showed multi-drug resistance, indicating that multi-drug resistant strains constituted the majority of clinical isolates. Huhuang decoction demonstrated significant attenuating effects against the multidrug resistance of P. multocida, with Scutellariae Radix identified as the primary active component responsible for this effect.

[Background] Aeromonas caviae is a widely distributed pathogen capable of infecting both humans and multiple animal species, posing significant threats to food safety and public health. [Objective] To investigate the pathogenic characteristics and antimicrobial resistance profile of swine-derived A. caviae, providing theoretical guidance for clinical prevention and control. [Methods] A dominant strain (designated HNHB) was isolated from the viscera of deceased piglets in a swine farm in Henan Province. The strain was identified by morphological observation, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and phylogenetic analysis based on the 16S rRNA gene. A Kunming mouse infection model was established to evaluate the pathogenicity of this strain based on the LD₅₀ value and histopathological changes, and the minimal inhibitory concentrations (MICs) of 14 antimicrobial agents against this strain were determined. [Results] The isolate HNHB was identified as A. caviae, with the 16S rRNA gene sequence similarity of 99.73% compared with the reference A. caviae strain (GenBank accession number: AP026375). Infection trials revealed the LD₅₀ of 6.24×10⁸ CFU/mL in Kunming mice. HNHB induced severe multiorgan lesions, including intestinal necrosis, hemorrhage, epithelial shedding, hepatomegaly, and nephromegaly. Antimicrobial susceptibility test results demonstrated that HNHB was sensitive to five antimicrobials including ceftazidime (MIC=0.32 μg/mL) and polymyxin (MIC=0.01 μg/mL) but resistant to florfenicol (MIC=128 μg/mL) and meropenem (MIC=160 μg/mL). [Conclusion] A strain of A. caviae, designated HNHB, was successfully isolated and identified from the visceral organs of deceased pigs. The isolated strain demonstrated pathogenicity in Kunming mice and exhibited a multidrug-resistant phenotype.

[Background] Staphylococcus aureus, the pathogen of bovine mastitis, has multidrug resistance and extensive drug resistance. Currently, most existing antibacterial drugs demonstrate limited efficacy and are prone to residue. Natural medicines have good inhibitory effects against drug-resistant bacteria. [Objective] To study the inhibitory activity and mechanism of quercetin against S. aureus causing bovine mastitis. [Methods] The K-B method was used to determine the sensitivity of S. aureus causing bovine mastitis to 15 antibiotics. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of quercetin against S. aureus causing bovine mastitis were determined by the broth microdilution method. The growth curve and bactericidal curve were established to analyze the inhibitory and killing effects of quercetin against S. aureus causing bovine mastitis. The chessboard method was adopted to determine the effects of quercetin combined with ampicillin, enrofloxacin, and doxycycline against S. aureus causing bovine mastitis. The mechanism of quercetin against S. aureus causing bovine mastitis was revealed through biofilm inhibition assay, nucleic and protein leakage assay, bacterial viability observation, quercetin accumulation analysis in bacteria, membrane protein conformation change assay, and molecular docking. [Results] The MIC and MBC of quercetin against S. aureus were 256 μg/mL and 512 μg/mL, respectively. Quercetin inhibited the growth of S. aureus and completely killed the bacteria within 12 h. The fractional inhibitory concentration indexes (FICIs) of quercetin with ampicillin, enrofloxacin, and doxycycline against S. aureus were 0.50, 0.75, and 0.50, respectively, which indicated synergistic or additive effects. Quercetin inhibited the biofilm formation of S. aureus (P < 0.05). Nucleic acid and protein leakage and bacterial viability analyses indicated that quercetin increased cell membrane permeability. The fluorescence intensity of quercetin in the bacterial suspension reduced (P < 0.05), which suggested that the concentration in bacterial cells gradually increased over time. Quercetin caused secondary and tertiary conformational changes of bacterial membrane proteins. Quercetin bound to tyrosyl-tRNA synthetase through van der Waals force and hydrogen bonding, which resulted in stable binding, thereby inhibiting enzyme activity. [Conclusion] Quercetin has inhibitory effect against S. aureus causing bovine mastitis by inhibiting the biofilm formation, enhancing the permeability of cell membrane, affecting the conformation of membrane proteins, and suppressing the activity of tyrosyl-tRNA synthetase.

[Background] China's pig industry is facing severe challenges from swine influenza virus (SIV), and the epidemic strains mainly include H1N1, H3N2 and H1N2, which continue to threaten the healthy development of the pig industry. [Objective] The objective of this study was to analyze the genetic evolution characteristics and systematically evaluate the pathogenicity of the circulating strains of SIV in Guangdong Province. [Methods] From 2021 to 2023, a total of 536 nasal swabs and lung lesion samples suspected of SIV infection were collected from 21 pig farms in Foshan, Zhaoqing, Qingyuan, Shaoguan and other prefecture-level cities in Guangdong Province, of which 45 samples were tested positive for SIV by quantitative RT-qPCR. The virus strains were isolated by culture with MDCK cells and then whole genome sequencing was performed. The obtained sequences were compared with those of the reference strains in GenBank and GISAID, and a phylogenetic tree was constructed to reveal the genetic evolution characteristics of the virus. [Results] Two SIV strains (SWSG1/21 and SWHRZ3/22) of EA H1N1 subtype were isolated, with HA and NA genes derived from the EA clade, PB2, PA, PB1, NP, and M genes derived from the Pdm/09 clade, and the NS gene derived from the TR H1N2 clade. In addition, two H1N2 subtype reassortant strains (SWLFT4/22 and SWHH5/23) were isolated. SWLFT4/22 was reassorted from strains of the NA H1N2 clade and Pdm/09 H1N1 clade, while SWHH5/23 involved the complex reassortment of strains from three different clades. The results of the pathogenicity test showed that all the four strains of SIV caused death in mice. Particularly, SWSG1/21 and SWLFT4/22 showed strong pathogenicity, leading to a mortality rate of up to 100%. [Conclusion] We analyzed the genetic evolution and pathogenicity of four SIV strains isolated from pigs in Guangdong Province and revealed the complex genetic recombination background and strong pathogenicity of local SIV, which provided a scientific basis for the formulation of swine influenza prevention and control strategies.

[Background] The Polyporaceae encompasses numerous species that exhibit highly diverse morphological traits; consequently, no fully satisfactory classification system has yet been established. Among its constituent genera, Trametes is the most taxonomically chaotic group within the family. [Objective] To expand the resource library of plant growth-promoting fungi, we isolated Trametes gibbosa SICAU-T41, a strain of poroid fungus, with plant growth-promoting effects from the rhizosphere soil of flue-cured tobacco in Huili County, Liangshan Prefecture, Sichuan Province. This study aimed to conduct whole-genome sequencing of strain SICAU-T41 to analyze its genomic structure, function, and phylogenetic position and mine its secondary metabolite gene resources. [Methods] Directed screening media and shake-flask culture were employed to isolate plant growth-promoting fungi, the plant growth-promoting characteristics of which were determined. Pot experiments and qPCR were then employed to evaluate the plant growth-promoting effects and colonization of the isolates. Whole-genome sequencing of strain SICAU-T41 was performed by Illumina and PacBio platforms. Gene prediction, functional annotation, prediction of secondary metabolite biosynthetic gene clusters (BGCs), and comparative genomic analysis were then conducted. [Results] A total of 78 fungal strains with distinct colony morphology were isolated and purified from the rhizosphere soil of flue-cured tobacco. Their abilities to solubilize inorganic phosphorus, produce indole-3-acetic acid (IAA), degrade cellulose, produce siderophores, and antagonize pathogens were measured. Strain SICAU-T41, exhibiting the strongest plant growth-promoting potential, was selected. This strain demonstrated the phosphorus-solubilizing capacity of 30.40 mg/L, IAA production of 48.10 mg/L, cellulose degradation zone diameter of 2.03 cm, and siderophore production (HD/CD ratio) of 2.00. Pot experiment results demonstrated that strain SICAU-T41 significantly promoted the growth of tobacco seedlings. Compared with the control (CK), inoculation of this strain increased the seedling height by 74.7%, root length by 14.4%, leaf number by 16.7%, leaf area by 48.4%, root number by 52.1%, aboveground fresh weight by 36.7%, and underground fresh weight by 82.6%. Compared with CK, the addition of SICAU-T41 significantly increased its colonization density in the rhizosphere soil of flue-cured tobacco, which reached 29.93×10³ copies/μL, a 123.36% increase over CK. Whole-genome sequencing revealed that the genome of strain SICAU-T41 had a length of 33.93 Mb, containing 10 142 predicted genes, including 9 843 protein-coding genes, with the G+C content of 56.89%. A total of 7 875, 3 444, 5 793, and 448 genes were annotated in the eggNOG, KEGG, GO, and CAZy databases, respectively. TCDB analysis predicted 1 129 transporters associated with various secretion systems. Furthermore, 22 secondary metabolite BGCs were predicted in the genome of strain SICAU-T41, involved in the synthesis of various natural products such as terpenoids, non-ribosomal peptides (NRPs), β-lactams, and ribosomally synthesized and post-translationally modified peptides (RiPPs). Comparative genomic analysis showed that the average nucleotide identity between strain SICAU-T41 and T. gibbosa reached 98.5%, confirming that SICAU-T41 was a strain of T. gibbosa. [Conclusion] The plant growth-promoting fungus T. gibbosa SICAU-T41, isolated from the rhizosphere soil of flue-cured tobacco, possesses multiple plant growth-promoting properties and significantly enhances tobacco seedling growth. Whole-genome sequencing and analysis of strain SICAU-T41 revealed the genetic basis underlying its plant growth-promoting mechanisms at the genomic level, while also enriching the genomic database of T. gibbosa.

[Background] Pigeon adenovirus (PiAdV), pigeon circovirus (PiCV), and pigeon herpesvirus (PiHV) are major pathogens that seriously jeopardize the health of pigeon flocks and often lead to coinfections, posing challenges for clinical differential diagnosis. [Objective] To establish a triplex TaqMan fluorescent quantitative PCR (real-time quantitative PCR, RT-qPCR) method for the simultaneous detection of PiAdV, PiCV, and PiHV. [Methods] We obtained the conserved regions by searching and comparing the genomes of PiAdV, PiCV, and PiHV published in GenBank. Primers and probes were designed based on the conserved region, and plasmid standards pMD19-T-PiAdV Ⅰ, pMD19-T-PiAdV Ⅱ, pMD19-T-PiCV, and pMD19-T-PiHV were prepared. The reaction systems were optimized and the standard curves were drawn to evaluate the specificity, sensitivity, and repeatability of the established assay. A triplex TaqMan RT-qPCR assay for simultaneous detection of PiAdV, PiCV, and PiHV was thus established. It was then used to test the clinical samples, and its clinical applicability was established. [Results] The standard curves of the triple TaqMan RT-qPCR assay for PiAdV, PiCV, and PiHV were y=–3.396x+43.210 (R²=0.998 3), y=–3.540x+40.540 (R²=0.999 4), and y=–3.280x+38.470 (R²=0.998 4), respectively, showing good linear relationships. The assay showed no cross-reaction with other common pigeon pathogens such as pigeon pox virus and pigeon rotavirus, as well as fowl adenovirus type 4, showcasing strong specificity. For PiAdV, PiCV, and PiHV, the assay showed the minimum detection limits of 10 copies/μL, 10 copies/μL, and 1 copy/μL, respectively, which are 100 to 1 000 times more sensitive than the conventional PCR method, indicating high sensitivity. The coefficient of variation was less than 2.0% in both intra-batch and inter-batch repeatability tests, demonstrating good repeatability and stability. The established triple TaqMan RT-qPCR assay for PiAdV, PiCV, and PiHV was used to detect 152 pigeon clinical samples and showed slightly higher positive rates (PiAdV 55.92%, PiCV 51.32%, and PiHV 40.13%) than the conventional PCR method, demonstrating good clinical applicability. [Conclusion] The established triple TaqMan RT-qPCR assay for the detection of PiAdV, PiCV, and PiHV has good linear relationships and high specificity, sensitivity, and repeatability. It can be used for rapid, efficient, and specific detection of PiAdV, PiCV, and PiHV in clinical samples, serving as an efficient technical method for early diagnosis of the three viruses as well as for the study of synergistic pathogenic mechanism and epidemiological investigation.

[Background] Proteus mirabilis is a major pathogen capable of causing infections in both humans and animals. Currently, the research on rapid detection techniques for this bacterium is scarce, posing challenges to early clinical diagnosis and targeted prevention and control efforts. [Objective] We developed two detection platforms based on recombinase polymerase amplification (RPA) and CRISPR/Cas12a systems, utilizing distinct protospacer adjacent motif (PAM) sequence strategies, to achieve rapid detection of P. mirabilis. [Methods] One-step and two-step detection platforms based on RPA and CRISPR/Cas12a systems were developed for P. mirabilis by targeting its conserved genes. The one-step platform simultaneously performs RPA and CRISPR/Cas12a reactions at 39 ℃, collecting fluorescence signals in a real-time manner. The two-step platform first conducts RPA at 39 ℃, and then transfers the CRISPR/Cas12a system to the bottom of the tube through brief centrifugation and continues the reaction at 39 ℃ to generate fluorescence signals. After optimization of the reaction conditions, we used the clinical isolates of P. mirabilis and other common bacterial samples to evaluate the sensitivity and specificity of both methods. [Results] Two detection platforms based on RPA and CRISPR/Cas12a were successfully established, both capable of detecting a minimum of 2.8 copies/μL of standard plasmid within 30 min. During sample pretreatment by thermal lysis, the one-step method achieved the sensitivity of 10⁶ CFU/mL, while the two-step method demonstrated the sensitivity of 10³ CFU/mL. After genomic DNA extraction from simulated samples, the sensitivity of the one-step and two-step methods reached 10² CFU/mL and 10¹ CFU/mL, respectively. Both methods demonstrated excellent detection ability across 30 clinical isolates and showed no cross-reactivity with other common pathogenic bacteria, showcasing good specificity. [Conclusion] We developed one-step and two-step RPA-CRISPR/Cas12a detection methods for P. mirabilis based on two distinct PAM sequence design strategies. These methods demonstrate rapid, sensitive, and specific detection performance, showing promising potential for on-site detection of P. mirabilis.

[Background] Porcine epidemic diarrhea (PED) remains a major disease in the swine industry in China and the globe. Therefore, understanding and monitoring the incidence and epidemic situation of this disease is of great significance for its prevention and control. [Objective] To establish a highly specific, sensitive, high-throughput, and highly automated chemiluminescent immunoassay for antibodies against porcine epidemic diarrhea virus (PEDV) using purified recombinant N protein of PEDV as the target antigen for monitoring PEDV epidemics or infections. [Methods] We constructed a recombinant plasmid by seamless cloning and induced the expression of N protein. The obtained protein was coupled with carboxy magnetic beads to form an immunomagnetic bead complex. After optimization of the reaction conditions, the PEDV antibody detection method was established. The specificity, sensitivity, reproducibility, and compliance rate of the established method were evaluated. [Results] The recombinant N protein was soluble and had good immunoreactivity. The optimal conditions for the established method were coupling pH 8.0, coupling protein amount of 80 μg, 20 μL 10% BSA as the blocking reagent, magnetic bead concentration of 0.25 mg/mL, sample dilution of 1:10, enzyme dilution of 1:15 000, incubation time of 10 min for the primary antibody, incubation time of 15 min for the secondary antibody, and substrate reaction time of 3 min. The established method showed higher sensitivity than ELISA and no cross-reactivity with 11 pathogen antibody-positive standard sera. The reproducibility test showed that the intra-batch and inter-batch coefficients of variation were 3.09%-8.80% and 4.87%-9.17%, respectively, which were both < 10%. The compliance test showed a positive compliance rate of 93.4%, a negative compliance rate of 99.2%, and an overall compliance rate of 98.2%. [Conclusion] The chemiluminescent immunoassay established in this study can be used to detect PEDV antibodies in clinical samples, track and understand the epidemic situation of PEDV, and provide a reference for subsequent reagent kit development.

[Background] Porcine parvovirus (PPV) is one of the major pathogens threatening the reproductive health of swine herds, causing tremendous economic losses to the pig farming industry and posing a serious threat to the quality and safety of porcine products. [Objective] To establish a rapid and sensitive detection method for PPV nucleic acid based on recombinase polymerase amplification (RPA) combined with lateral flow dipstick (LFD) assay. [Methods] We designed specific primer pairs based on the conserved sequences of the NS1 gene in PPV. Then, we developed a RPA-LFD method for the detection of PPV by optimizing the reaction conditions. [Results] The RPA-LFD method can detect PPV nucleic acid within 20 min at 39 ℃, with a limit of detection of 10 copies/μL. The experimental results showed that only PPV was positively detected by this method, whereas no cross reaction with porcine circovirus type 2, pseudorabies virus, swine fever virus, porcine reproductive and respiratory syndrome virus or foot-and-mouth disease virus was observed. In addition, the porcine tissue samples artificially polluted with PPV and the simulated clinical samples were detected by RPA-LFD and traditional PCR, and the results of the two methods were consistent. [Conclusion] The developed RPA-LFD method does not rely on a thermal cycler and it is simple to operate and highly sensitive and specific, providing an effective strategy for the on-site rapid detection of PPV nucleic acid.