Viruses are the most abundant biological entities on Earth and widely exist in various ecosystems. They play a role in regulating microbial community composition, influencing microbial evolution, participating in element biogeochemical cycles, and even causing plant and animal diseases. Soil is an important reservoir of viruses. Due to the limitations of soil heterogeneity, adsorption of soil colloids, and lack of research and analysis methods, the research on soil viruses lags far behind that on viruses in marine or other aquatic ecosystems. In recent years, the rapid development of molecular biology and the gradual improvement of ecological theory have promoted the research on soil viruses, researchers gradually paid attention to the function of viruses in soil ecosystem. In this paper, we summarized the research status and progress of soil viruses in terms of the types, research methods, and ecological functions. On this basis, the future development trends and key research directions of soil viruses were prospected in this paper. This review can deepen people’s understanding of soil viruses and provide scientific reference for the research on soil viruses.

This article explores the dynamic regulation of fluid shear stress on the biofilm formation of Streptococcus mutans, aiming to elucidate the pathogenic mechanism of this bacterium and provide references for the prevention of dental caries. S. mutans is a major cariogenic bacterium in the oral cavity, and it destroys the tooth tissue by forming biofilms and producing acids by metabolism. Studies have shown that fluid shear stress can regulate the physical structure, secretion of extracellular polymeric substances, adhesion, and quorum sensing of biofilms, thereby affecting the pathogenicity. This study provides a theoretical basis for deeply understanding the ecological adaptability of pathogens in fluid environments and the development of new intervention measures in clinical practice.

Currently, the drugs available for treating fungal infections show limited number and off-target effects. Thus, there is an urgent need to develop new antifungal drugs. Fungal apoptosis-like cell death (ALCD) is a cell death phenomenon that occurs during the normal development stage of organisms. This article summarizes the characteristics, signaling pathways, and key factors involved in fungal ALCD and introduces the natural and synthetic drugs that can induce fungal apoptosis. The natural drugs include lipid peptides, farnesol, statins, and alkaloids from microorganisms, organic acids and essential oils from plants, and melittins from insects. Furthermore, this article establishes the basic molecular landscape of drug-induced fungal ALCD. This article provides a theoretical basis for formulating a new strategy for resisting pathogenic fungi and developing targeted antifungal drugs.

Brucellosis is a zoonotic disease caused by Brucella. Brucella can replicate within various host cells and evade the host immune system, which underpins its pathogenicity. Rough Brucella strains, characterized by the absence or inadequacy of the O-antigen chain, exhibit incomplete lipopolysaccharide structures, precluding the production of anti-lipopolysaccharide antibodies. This distinguishing feature facilitates differential diagnosis and has attracted considerable attention. Rough Brucella can invade and manipulate the host immune system via low endotoxin activity, lysosomal escape, and resistance to complement attacks. Nevertheless, comprehensive investigations into the host immune responses, rough Brucella-host interaction mechanisms, and virulence gene functions of rough Brucella remain scarce. This article elucidates the intracellular parasitism process of rough Brucella and the molecular mechanisms underlying their interactions with hosts, with the aim of offering insights for the development of effective vaccines against rough Brucella.

Hydroxylamine, as an important intermediate product in the nitrogen cycle, connects ammonia oxidation and nitrite oxidation, influencing the velocities and directions of processes like ammonia oxidation, nitrite oxidation, and denitrification. Because of the close associations with the generation of N₂O through enzymatic reactions and self-decomposition or reactions with other substances, hydroxylamine has become a focus and hotspot of research. This paper summarized the generation and transformation of hydroxylamine in autotrophic and heterotrophic ammonia oxidation, the key role of hydroxylamine in the nitrogen cycle, and the promoting effect of hydroxylamine on N₂O emissions. It analyzed the processes of autotrophic and heterotrophic ammonia oxidation and their enzymatic differences, aiming to provide a theoretical reference for in-depth research on the role of hydroxylamine in the microbial nitrogen cycle and for the research and development of measures to reduce N₂O emissions and protect the atmospheric environment.

Saccharomyces cerevisiae is a classic model organism for studying the biochemical mechanisms of eukaryotic cells. Eukaryotes have three main types of RNA polymerases: RNA polymerase I (RNAPI), RNA polymerase II (RNAPII), and RNA polymerase III (RNAPIII). Among them, RNAPIII has the most complex structure, consisting of 17 subunits, and it is primarily responsible for the synthesis of transfer RNA (tRNA). Compared with RNAPII consisting of 12 subunits, RNAPIII contains a unique heterotrimer Rpc82/31/34 and a heterodimer Rpc53/37 which is homologous to the counterpart of RNAPI. This paper reviews the structures and functions of the specific heterotrimer and heterodimer in RNAPIII, aiming to lay a theoretical foundation for further studies on the modification mechanisms and assembly processes of specific subunits of RNAPIII in S. cerevisiae.

Transcription factors are essential for the survival of bacteria, and revealing their functions is conducive to the development of antimicrobials, industrial applications, and environmental protection. At present, there are many methods and strategies for the research on transcription factors, which have been developed rapidly in the fields of bioinformatics and molecular biology. This paper summarizes the methods for identifying unknown transcription factors, unveiling the regulatory functions of transcription factors, constructing regulatory networks of transcription factors, and validating the regulatory genes of transcription factors. This review aims to provide new strategies and new ideas for the analysis and verification of the regulatory functions of bacterial transcription factors.

The aggravating soil-borne diseases threaten the production of a variety of crops and undermine the sustainable development of agriculture. Streptomyces-based bio-agents have been widely explored as an environmentally safe and economically durable biocontrol resource. However, the biocontrol efficacy and influencing factors require further optimization. [Objective] Here we conducted a meta-analysis to evaluate the efficacy of Streptomyces-based bio-agents in controlling the two common soil-borne diseases and identify the key influencing factors. [Methods] The relevant articles were retrieved from Web of Science and CNKI with the keywords “Streptomyces” and “Fusarium wilt”, “Streptomyces” and “bacterial wilt”, “Streptomyces” and “Fusarium oxysporum”, and “Streptomyces” and “Ralstonia solanacearum (or Pseudomonas solanacearum)”. The articles containing comparable treatment groups (with Streptomyces application) and control groups (without Streptomyces application), disease incidence, sample sizes, and mean values were systematically selected. Finally, 76 articles (113 groups) on Streptomyces-based bio-agents for Fusarium wilt control and 19 articles (28 groups) on those for bacterial wilt control were obtained. [Results] After the application of Streptomyces-based bio-agents, the disease incidence of Fusarium wilt decreased from 75.58% to 24.49% (average control efficacy of 67.60%), and that of bacterial wilt decreased from 73.75% to 19.83% (average control efficacy of 73.11%). The soil density of Streptomyces, the final concentration ratio of Streptomyces to pathogen in soil, and climate types were vital for the biocontrol performance of Streptomyces-based bio-agents. Moreover, Streptomyces at the final concentration of 10⁷ CFU/g demonstrated the best biocontrol performance on both diseases. The biocontrol effect of Streptomyces on Fusarium wilt was better when the final concentration ratio of Streptomyces to pathogen was 1:1, whereas the biocontrol effect on bacterial wilt disease was better when the ratio was 10:1 or 100:1. Notably, Streptomyces demonstrated enhanced biocontrol effects on the both diseases in tropical monsoon climate regions. [Conclusion] The application dose of Streptomyces-based bio-agents in soil-borne disease management should be adjusted according to the species and concentration of the pathogen. In addition, it may be better to apply Streptomyces-based bio-agents in tropical monsoon climate regions.

[Objective] Listeria monocytogenes (Lm) is a Gram-positive foodborne pathogen causing human and animal listeriosis with high case fatality rates (20%-30%). Lm can grow at low temperatures, which brings great risks to food safety and human health. Exploring the mechanism of Lm growth at low temperatures will provide a theoretical basis for the formulation of measures to control Lm growth at low temperatures and the development of low-temperature growth inhibitors. Studies have shown that LisK/R, a two-component signal transduction system (TCS) in Lm, plays a role in regulating the growth of Lm at low temperatures, while the specific mechanism remains unclear. This study aims to reveal the mechanism of LisK/R in regulating the growth of Lm at low temperatures. [Methods] With the Lm strain LM201 isolated from foodstuff as the parental strain, we constructed three strains: ΔlisR (RR-deleted mutant of LisK/R), ΔlisRc (complementary strain of ΔlisR), and ΔlisKc (complementary strain of ΔlisK, which was constructed before this study in our laboratory). The growth curves of these strains were determined at 4 ℃. RNA-Seq was performed for ΔlisK and the parental strain growing at 4 ℃, and the obtained data were analyzed. We assayed the swarming area diameter on soft agar plate, flagellar biosynthesis by transmission electron microscopy, and transcriptional levels of flagellar genes by RT-qPCR for all the strains at 4 ℃. [Results] The growth of deletion mutants was slower than that of the parental strain (P<0.05), and the growth of complementary strains was consistent with that of the parental strain at 4 ℃. The RNA-Seq data showed that compared with that in the parental strain, the expression of flagellar genes in ΔlisK was up-regulated (P<0.05). At 4 ℃, the swarming area diameters of deletion mutants on soft agar plates were bigger than those of the parental strain and complementary strains (P<0.05); the deletion mutants had more flagella, while the parental and complementary strains had fewer flagella; the expression of flagellar genes in the deletion mutants was higher than that in the parental and complementary strains (P<0.05). [Conclusion] At 4 ℃, the LisK/R mutants with slow growth had more flagella, while the parental strain with rapid growth had fewer flagella. The results indicate that LisK/R inhibits the expression of flagellar genes in Lm to reduce flagellar production, and the energy is used to promote Lm growth at low temperatures. The specific molecular regulatory mechanism of LisK/R needs further study. This study provides new information for elucidating the mechanism of Lm growth at low temperatures.

Allergic asthma is a prevalent chronic inflammatory disease characterized by airway hyperresponsiveness and airway inflammation, often triggered by inhalant allergens such as pet dander and pollen. [Objective] Studies have revealed that helper T cells (Th cells) play a crucial role in the immune regulation of allergic asthma. Particularly, Th2 cells exacerbate airway inflammation by secreting cytokines such as interleukin (IL)-4 and IL-5, which promote the proliferation of eosinophils and mast cells. Additionally, the activation of Th17 cells is closely related to the inflammatory response in allergic asthma. Therefore, modulating the balance of Th1/Th2 and Th17/Treg cells has become an effective strategy for treating allergic asthma. This study explores the pathogenesis of allergic asthma and novel therapeutic approaches. [Methods] We constructed an engineered bacterium EcN ^canf-1 specifically expressing the dog allergen Canf-1. Using the pBAD promoter, we controlled the expression of Canf-1 in the lungs of mice. [Results] EcN ^canf-1 exerted immunomodulatory effects by regulating pulmonary cytokine expression profiles, specifically downregulating the expression of pro-inflammatory cytokines including IL-6, IL-5, and IL-13, while upregulating the expression of anti-inflammatory mediators such as interferon-γ (IFN-γ), transforming growth factor-β (TGF-β), and IL-10, thus alleviating allergic symptoms in the lungs. EcN ^canf-1 demonstrated remarkable efficacy in suppressing the pathological overexpression of Th2 and Th17 lymphocyte subsets under allergic conditions, while enhancing the functions of Th1 and regulatory T cells. Furthermore, EcN ^canf-1 significantly reduced pulmonary mast cell infiltration, attenuated vascular permeability, and mitigated allergen-induced hypothermia and airway constriction. These findings demonstrated that EcN ^canf-1 played a pivotal role in immune homeostasis regulation and represented a promising therapeutic candidate for allergic pulmonary inflammation through its multifaceted modulation of immunological pathways. [Conclusion] EcN ^canf-1 provides a new therapeutic avenue for allergic asthma by modulating the expression of immune cells and cytokines. This not only offers new insights into the immune mechanisms of allergic asthma but also provides a scientific basis for the development of new targeted biological treatment strategies.

[Objective] To isolate and screen the salt-tolerant strains with good plant growth-promoting effect from the saline-alkali soil of the Yellow River Delta, thus providing strain resources for the efficient cultivation of crops in saline-alkali soil. [Methods] Bacillus strains were isolated by the dilution coating method, and the strains with good plant growth-promoting effects were further selected by the seed soaking test. The growth-promoting characteristics of the selected strains were measured, and the growth-promoting effects of the strains on Sesbania cannabina under salt stress was evaluated by pot experiments. The strain with the strongest plant growth-promoting effect was identified based on morphological characteristics, physiological and biochemical characteristics, and molecular biological evidence. Through the whole genome sequence analysis, the genes related to the plant growth-promoting function were discovered. [Results] A total of 60 Bacillus strains were isolated, from which strains M4, M5, B5, L3, and Q17 were screened out by the seed soaking test. These strains showed robust plant growth-promoting properties, being capable of solubilizing inorganic phosphorus, solubilizing potassium, and producing inole-3-acetic acid. The pot experiment results showed that under normal culture conditions and under low salt stress (NaCl concentration of 100 mmol/L), inoculation with Bacillus increased the plant height, maximum leaf area, stem dry weight, and root dry weight of S. cannabina seedlings (P<0.05). Under high salt stress (NaCl concentration of 200 mmol/L), the fresh and dry weights of stem and leaves of S. cannabina seedlings were increased by inoculation with five strains of Bacillus (P<0.05). In addition, inoculation with Bacillus enhanced the activities of catalase, superoxide dismutase, and peroxidase while reducing the content of malondialdehyde in the leaves of S. cannabina seedlings (P<0.05). Strain M4 with the strongest plant growth-promoting effect was identified as Bacillus thuringiensis. [Conclusion] All the five isolates have various plant growth-promoting properties, being capable of promoting the growth of S. cannabina seedlings under salt stress and alleviating the inhibitory effect of salt on the seedlings. Strain M4 with the robust plant growth-promoting effect is identified as B. thuringiensis, and it has the potential to be developed as plant growth-promoting bacterial fertilizer for saline-alkali soil.

[Objective] To isolate and identify a strain of foot-and-mouth disease virus (FMDV) from bovine oropharyngeal fluid (OPF) and subsequently analyze the amino acid mutations in antigenic proteins and the proliferative properties of the isolated virus. [Methods] RT-PCR and sequencing were performed to identify the FMDV-positive OPF samples. The FMDV strain was isolated from bovine OPF samples by cell inoculation and identified by indirect immunofluorescence assay. The whole genome sequence of the strain was obtained by fragment amplification and sequencing. The amino acid sequences were compared to reveal the amino acid mutations in the antigenic proteins of the strain. The proliferative properties of the isolated FMDV strain were analyzed by the plaque forming assay and one-step growth curve. [Results] A FMDV strain O/FMDV/OP/2022/B was successfully isolated from bovine OPF, belonging to serotype O and Ind-2001 lineage. Amino acid mutations were identified in the antigenic proteins VP1, VP2, and VP3 of O/FMDV/OP/2022/B. In addition, the plaque of O/FMDV/OP/2022/B in BHK-21 cells was smaller than that of the vaccine strain O/FMDV/HN/93. The one-step growth curves showed that O/FMDV/OP/2022/B had significantly lower replication titer than O/FMDV/HN/93 in BHK-21 cells. [Conclusion] A strain of FMDV was isolated from bovine OPF, and it displayed amino acid mutations in antigenic proteins and had lower proliferative capacity than the vaccine strain O/FMDV/HN/93. This study contributes to a better understanding of the mechanism of persistent FMDV infection in the bovine oropharynx and provides a reference for the prevention and control of FMD.

[Objective] To extract and purify extracellular polysaccharides (EPS) from Sphingomonas echinoides 1K04342 and investigate the ameliorating effects and mechanisms of the purified EPS on glucose and lipid metabolism disorders induced by oleic acid (OA) in human hepatoma cells (HepG2). [Methods] A response surface methodology was employed to optimize the ultrasound power, ultrasound time, and incubation time for maximizing the EPS yield of S. echinoides 1K04342 from ultrasound-assisted extraction. The crude EPS underwent deproteinization and was further purified through a DEAE-Sepharose Fast Flow anion exchange column. The effects of the purified EPS on the proliferation, glucose uptake, and α-glucosidase inhibition and pancreatic lipase inhibition rates in HepG2 cells were evaluated. The most effective EPS component for mitigating OA-induced glucose and lipid metabolism disorders in IR-HepG2 cells was selected for in-depth physicochemical and bioactivity analyses. [Results] The optimal EPS extraction parameters were as follows: ultrasound power at 59.39 W, extraction for 42.08 s, and incubation for 9.24 h, under which the estimated yield of EPS reached 2.13 g/L. Under the adjusted parameters (60 W, 42 s, and 9.2 h), the EPS yield was 2.10 g/L, which was statistically consistent with the theoretical value (2.13 g/L). The optimal component for ameliorating glucose and lipid metabolism disorders in IR-HepG2 cells was EPS-2, which showed the total sugar content of 74.3%, with a monosaccharide profile comprising fucose, rhamnose, arabinose, galactose, glucose, and mannose. EPS-2 alleviated OA-induced glucose and lipid metabolic disorders in IR-HepG2 cells. EPS-2 reversed OA-induced decreases in cell viability. EPS-2 may activate the IRS-1/PI3K/AKT pathway, up-regulating the protein levels of GSK3β and FoxO1 and down-regulating the mRNA and protein levels of PEPCK and G6Pase, thereby mitigating glucose metabolism disorders. EPS-2 may regulate OA-induced lipid metabolism disorders by activating the AMPK/ACC1/SREBP-1C pathway, lowering the triacylglycerol (TG), total cholesterol (TC), and low-density lipoprotein (LDL) levels, and increasing the high-density lipoprotein (HDL) level. [Conclusion] EPS-2 derived from S. echinoides 1K04342 could effectively ameliorate glucose and lipid metabolism disorders in IR-HepG2 cells.

[Objective] To explore the antifungal activity and inhibitory mechanism and identify the main antagonistic substances of the crude extract of Pseudomonas protegens FD6 in fermentation. [Methods] The plate confrontation test was employed to screen the optimal medium, extractant, and solvent and determine the antifungal activity stability of the crude extract. The main antifungal substances in the crude extract were identified by MALDI-TOF/TOF mass spectrometry, and the antifungal spectrum of the crude extract was explored. The inhibition mechanism of the crude extract against Botrytis cinerea was investigated by mycelial morphology observation, spore germination test, PI staining, and conductivity determination. [Results] The crude extract of FD6 in the potato juice medium without any carbon source displayed the strongest antifungal activity. The methanol-solved crude extract obtained with ethyl acetate as the extractant had the strongest inhibitory activity. The main antifungal substance of the crude extract was identified as a cyclic lipopeptide, orfamide A, based on mass spectrometry data. In addition, the crude extract displayed broad-spectrum inhibitory activities against Phomopsis amygdali, Fusarium graminearum, Colletotrichum gloeosporioides, and Monilinia fructicola. The antifungal activity of the crude extract was stable after treatment at 121 ℃ for 20 min, within the range of pH 4.0-10.0, and with protease K and trypsin. The mycelial morphology of B. cinerea became abnormal after treatment with the crude extract. The treatment with 2.0 mg/mL crude extract completely inhibited the conidial germination of B. cinerea, caused a 50% damage of the cell membrane, increased the conductivity, and led to leakage of intracellular nucleic acids and proteins. [Conclusion] The metabolites produced by P. protegens FD6 have broad-spectrum and stable antifungal activities, serving as lead compounds for exploration of new green fungicides.

[Objective] To investigate the antibiotic resistance and virulence of pet-derived Klebsiella pneumoniae in some areas of Guangdong Province. [Methods] Fecal swabs were collected from dogs and cats for strain isolation, and 16S rRNA and khe genes were amplified by PCR to identify the strains. The sensitivity of K. pneumoniae isolates to 17 antibiotics was determined by the agar diffusion method. PCR was employed to detect resistance genes to β-lactams (bla_SHV, bla_CTX, and bla_TEM), carbapenems (bla_KPC and bla_NDM), aminoglycosides (rmtB), quinolones (qnrS and oqxAB), sulfonamides (Sul1 and Sul2), amphenicols (floR), tetracyclines (tet(A)), and fosfomycin (fosA3) and some virulence genes (rmpA, maga, fimH, mrkD, uge, WabG, kfu, Aerobactin, and ureA). [Results] A total of 126 strains of K. pneumoniae were isolated from 428 fecal samples, with an isolation rate of 29.4%. The 126 isolates had high resistance rates to amoxicillin (75.40%), ampicillin (73.81%), and cotrimoxazole (61.90%). They were moderately sensitive to ceftazidime, amikacin, apramycin, and enrofloxacin, and they were sensitive to tigecycline, colistin, and meropenem. The detection rate of the resistance gene oqxAB was the highest, which was 86.51%, followed by those of the β-lactam resistance gene bla_SHV (73.81%) and the tetracycline resistance gene tet(A) (52.68%). Other resistance genes were detected to varying degrees (0.79%-46.03%) and the carbapenem resistance gene bla_KPC, colistin resistance gene mcr-1, and aminoglycoside resistance gene rmtB were not detected. Among the virulence genes, the urease gene urea, lipopolysaccharide-related gene uge, and fimbria-related gene fimH showed the detection rates of 100.00%, 95.54%, and 91.07%, respectively. Other virulence genes were detected to varying degrees (2.70%-8.90%), while the capsule-related gene magA and the fimbria-related gene mrkD were not detected. [Conclusion] In some areas of Guangdong Province, the antibiotic resistance of pet-derived K. pneumoniae is serious, but its pathogenicity is relatively weak. Monitoring of its drug resistance and pathogenicity should be strengthened. In addition, antibiotics should be strictly managed and rationally used in the clinical practice, so as to avoid the generation and dissemination of multidrug resistant strains of K. pneumoniae.

[Objective] To delineate the fungal diversity characteristics and disparities in the rhizosphere soil of naked barley from fields exhibiting different root rot incidences, thereby informing targeted and effective disease management strategies. [Methods] Rhizosphere soil samples of naked barley were collected from the healthy field and the fields with root rot incidences of 5%, 10%, 15%, and 20%, respectively. The 18S rRNA gene of fungi from the samples was amplified, and high-throughput sequencing was conducted on the Illumina-MiSeq platform. Following quality control, classification, and annotation, the data were analyzed for fungal diversity from various perspectives and taxonomic levels. [Results] The healthy sample and the sample with the root rot incidence of 5% had the highest fungal diversity, while the sample with the incidence of 10% exhibited the lowest diversity. Additionally, co-occurrence network analysis revealed more complex species interactions in the healthy sample and the sample with the incidence of 5%. The root rot incidence had a negative correlation with the relative abundance of Ascomycota but a positive correlation with the relative abundance of Basidiomycota and Glomeromycota. Dominant fungal classes were Agaricomycetes, Chytridiomycetes, and Sordariomycetes. Significant variations in the distribution of fungal groups with high average relative abundance were noted across samples. Specially, the healthy sample had the highest relative abundance of Eurotiales and the lowest relative abundance of Glomerales, which were converse in diseased samples. Arthrodermataceae was significantly enriched in the healthy sample, while Sordariaceae, Myxotrichaceae, and Olpidiaceae were preferentially associated with the samples exhibiting incidences of 10%, 15%, and 20%, respectively. At the genus and species levels, the composition of dominant fungal communities in the healthy sample and the sample with the incidence of 5% was similar, and it was similar in the samples with incidences of 10%, 15%, and 20%. FUNGuild predicted a decrease in the relative abundance of plant pathogens and an increase in the relative abundance of saprotrophs with the increase in disease incidence. [Conclusion] The progression of naked barley root rot is intricately linked to the disruption of the equilibrium within the rhizosphere fungal community. In the context of precision management of naked barley root rot, it is imperative to regulate and sustain the balance of the abundance of dominant fungal taxa in the rhizosphere.

[Objective] Conventional culture methods can merely uncover a fraction of microbial diversity. A vast number of microorganisms remain unculturable under laboratory settings. The advent of in-situ culture technology offers a key solution to this predicament. This study endeavors to innovate the in-situ culture technology, explore microorganisms under hitherto unknown culture conditions, and probe into their potential applications within the realm of uncultured microorganisms. [Methods] PCR tubes were employed as the core of the device, and the idea of sorting prior to culture was introduced. Microorganisms were separated from the environment by means of polymer membranes for independent culture. The efficacy of the device was validated through the pure culture of Escherichia coli. Moreover, this device was applied to in-situ culture in diverse environments such as soil, sewage, and mountain spring water. [Results] The abundance of E. coli increased significantly in the pure-culture device. When the concentration of the polymer membrane reached 15%, the encapsulation effect effectively prevented the escape of E. coli. The co-culture experiments further corroborated this finding. In the in-situ culture experiments, the device designed in this study successfully cultured single-cell microorganisms from sewage, soil, and mountain spring water samples. Sequencing results indicated that this device could culture species recalcitrant to be cultured in the laboratory. Comparison with the NCBI database verified that new species were successfully cultured, which demonstrated the effectiveness of the culture device in various environments. [Conclusion] The culture method designed in this study is suitable for single-cell microbial culture, enrichment of specific microbial communities, and co-culture of multiple microorganisms. The device can isolate and culture richer and more microorganisms than conventional culture methods. This new technology not only isolates and cultures more microorganisms but also manages to culture those previously unculturable under laboratory conditions. It holds great significance for microbiological and ecological research.

[Objective] To study the response relationship of the biological properties of bacterial communities to long-term climate change. [Methods] The loess (YL, PL, and RL) and paleosol (YS, PS, and RS) sequences, which have persisted for approximately 500 000 years at Shimao (Y), Potou (P), and Renjiapo (R), were sampled. The structures and functions of the soil bacterial communities were predicted by bioinformatics approaches, including high-throughput sequencing and FAPROTAX. [Results] The physical and chemical characteristics associated with the loess-paleosol alternation in the three regions reflected the shifts in climatic conditions, including dry, cold, warm, and wet phases during soil development. Over this period, the climate at Renjiapo was characterized by the highest temperatures and precipitation levels, with strong influences from summer winds. Shimao experienced the driest conditions and the lowest temperatures. Potou exhibited the intermediate climatic conditions between Renjiapo and Shimao. The abundance and diversity indices of the bacterial communities in Shimao were higher than those in Potou and Renjiapo. Across all the three regions, the dominant bacterial phyla were Proteobacteria, Actinobacteria, and Acidobacteria although their relative abundance varied significantly. The bacterial communities in the loess and paleosol layers of Shimao showed greater compositional similarity, forming a network structure driven by synergistic interactions. The ecological functions of the bacterial communities in the loess-paleosol sequences were primarily associated with carbon, nitrogen, and sulfur cycling. Carbon and nitrogen cycling was weakly expressed in Shimao, while nitrogen cycling was more prominent in Potou. Renjiapo exhibited the most pronounced carbon cycling. [Conclusion] Soil bacterial communities in warm and humid climates tend to exhibit more complex network structures and greater functional diversity. In contrast, bacterial communities in dry and cold climates are characterized by similar composition and synergistic patterns, which enable these communities to adapt to harsh conditions by increasing their abundance and diversity, compensating for nutrient limitations associated with reduced carbon and nitrogen cycling.

[Objective] Stenotrophomonas can promote the growth of plants and aquatic algae. However, limited studies have focused on the bacteriophages infecting Stenotrophomonas. In this study, we isolated bacteriophages infecting Stenotrophomonas from water and studied their biological and genomic characteristics, aiming to improve our understanding of the ecological functions of Stenotrophomonas and its bacteriophages. [Methods] The double-layer plate method was employed to isolate bacteriophages infecting S. indicatrix EB12 from lake water. The biological characteristics, whole genome sequence characteristics, gene functions, phylogenetic relationship, and protein structures of the isolates were analyzed. [Results] Transmission electron microscopy showed that two isolates Ste-X and Ste-D were short-tailed bacteriophages. The incubation period of both strains was 20 min. Ste-X and Ste-D showed the optimal multiplicities of infection being 1 000 and 10, burst sizes of 28.8 PFU/cell and 131.1 PFU/cell, temperature tolerance ranges of 20-60 °C and 20-70 °C, and pH tolerance ranges of pH 4.0-13.0 and pH 3.0-12.0, respectively. Ste-X and Ste-D were deactivated by UV irradiation for 60 min and 120 min, respectively. Their host ranges were narrow. The genomes of both strains showed the lengths of 39 429 bp, with the G+C content of 57.80% and 57.85%, respectively, and both of them contained 65 open reading frames (ORFs). The genomes of the two strains contained only three different bases. However, their homology with other bacteriophages was low, which suggested that the two strains were novel short-tailed bacteriophages. The structures of the proteins encoded by ORF34 and ORF52 were predicted. Result showed thant the proteins encoded by ORF34 were acidic, stable, and hydrophilic, containing two conserved domains, ChtBD3 and PHA00661, with the three-dimensional structures dominated by random coils. The proteins encoded by ORF52 were alkaline, unstable, hydrophilic, with no conserved domain and the three-dimensional structures dominated by α-helixes. [Conclusion] Two novel strains of short-tailed bacteriophages with high genome similarity but distinct biological characteristics were isolated from water.

[Objective] Using Sphingomonas paucimobilis as the starting strain, a high-yield gellan gum-producing engineered strain was constructed through metabolic engineering, and fermentation process optimization was performed, providing both theoretical support and technical foundations for the efficient biosynthesis of gellan gum with this bacterium. [Methods] A CRISPR-Cas9-based gene editing system was developed for S. paucimobilis, and subsequently employed to genomically integrate two key gellan gum biosynthesis genes: the regulatory protein gene (gelA) and the β-1,4-glucuronosyltransferase gene (gelK), both under the control of constitutive promoters. Building upon this foundation, fermentation parameters including carbon source, nitrogen source, pH, and dissolved oxygen were systematically optimized through single-factor experiments. [Results] The engineered strain FMME-GG08 achieved a gellan gum yield of 10.8 g/L in shake-flask cultivation, representing a 130.2% enhancement over the parental strain. Following fermentation process optimization, the production level reached 20.1 g/L in 15 L scale bioreactors, with a sucrose conversion efficiency of 0.50 g/g. [Conclusion] This study not only successfully constructed a high-yield gellan gum-producing strain and established an efficient fermentation process, providing a reliable technical solution for industrial production, but also developed a genetic editing strategy that serves as an important reference for metabolic engineering of non-model microorganisms to produce other high-value exopolysaccharides.

The vpa1443-vpa1445 gene cluster (mfpABC) encoding the membrane fusion protein (MFP) is hypothesized to be involved in the biofilm formation of Vibrio parahaemolyticus (Vp). The gene mfpA (vpa1445) encodes a Ca²⁺-binding extracellular protein containing a repeats-in-toxin (RTX) domain, while its function is still under exploration. [Objective] To study the influences of mfpA mutation on the biofilm formation and motility of Vp. [Methods] The single mutants of three genes in the mfpABC gene cluster were constructed, and the Vp motility was compared between the three mutants and the wild type. Furthermore, the influences of mfpA mutation on bacterial motility and biofilm formation were analyzed in detail, and the expression of related genes was analyzed by RT-qPCR. Moreover, the cytotoxicity of ΔmfpA to HeLa cells was investigated. [Results] The mutation of mfpA significantly reduced the swimming and swarming motility of Vp. Crystal violet staining and scanning electron microscopy results showed that the mutation of mfpA enhanced the biofilm formation and increased the content of extracellular polysaccharides and proteins. RT-qPCR confirmed that the expression levels of flagellar genes were downregulated, while those of extracellular polysaccharide synthesis-related genes were upregulated in ΔmfpA. The cytotoxicity of ΔmfpA significantly decreased compared with that of wild type. [Conclusion] The mutation of mfpA can affect the expression of genes associated with flagella and extracellular polysaccharides to reduce the motility, enhance the biofilm formation, and attenuate the cytotoxicity of Vp.

[Objective] To improve the overall quality of upper tobacco leaves. [Methods] We isolated a hemicellulase-producing bacterium from tobacco soil and identified it as Actinacidiphila bryophytorum. After optimizing the culture conditions of this strain, we prepared the crude enzyme liquid, which was sprayed on upper tobacco leaves. The physical properties and chemical composition of the treated tobacco leaves were then evaluated. [Results] The optimal fermentation conditions for the strain were an inoculation rate of 4%, pH 6.0, and incubation at 30 ℃ for 36 h. Enzyme preparations of different concentrations (50, 100, 150, 200 and 250 U/mL) were then sprayed on the surface of upper tobacco leaves, and the appearance quality, conventional chemical composition, lignocellulose content, and sensory scores of the tobacco leaves were evaluated. The results indicated that treatment of the enzyme preparation at 150 U/mL performed better than other treatments. Compared with the control group, the 150 U/mL treatment increased the overall score for the appearance quality (maturity, color, characteristics, and structure) of tobacco leaves by 5.15 points. Additionally, this treatment increased the total sugar content and reducing sugar content by 29.87% and 35.77%, respectively. Meanwhile, it decreased the content of nicotine, cellulose, hemicellulose, and lignin by 16.10%, 16.37%, 20.22%, and 17.13%, respectively. Furthermore, the aroma characteristics were enhanced, and off-flavors were reduced. [Conclusion] The enzyme preparation produced by the fermentation of A. bryophytorum S2 improves the appearance quality, increases the soluble total sugar and reducing sugar content, and reduces the lignocellulose content of tobacco leaves, thus improving the overall quality of tobacco leaves.

[Objective] Yersinia enterocolitica is the primary pathogenic agent of yersiniosis, and it is one of the major foodborne pathogens worldwide. The purpose of this study is to isolate and identify a virulent phage against Y. enterocolitica and explore its application potential. [Methods] A novel virulent phage, named Yersinia phage Yen-yong1 (abbreviated as Yen-yong1), was isolated from the sewers of Lulin Market in Beilun District, Ningbo City, Zhejiang Province, China, by the double-layer agar method with Y. enterocolitica CICC 10869 as the indicator host. The morphology, host range, genome sequence, gene functions, phylogenetic position, physical and chemical tolerance, and biofilm removal ability of Yen-yong1 were studied. Additionally, the sterilization effect of Yen-yong1 on Y. enterocolitica in raw pork slices was evaluated. [Results] Yen-yong1 clarified the turbid CICC 10869 culture within 1.5 h. Yen-yong1 was Siphovirus-like, consisting of an elliptical head with a length of (83.32±0.15) nm and a width of (59.57±2.28) nm and a long tail with a length of (168.04±3.21) nm. The double-stranded DNA genome of Yen-yong1 was 51 321 bp, with the G+C content of 47.80% and 93 open reading frames (ORFs). No known lysogeny-associated genes, resistance genes, or virulence genes detrimental to application were found in Yen-yong1. The highest nucleotide sequence similarity between Yen-yong1 and the other phages in public databases was 20.64%, which was <70.00% (the threshold to define a genus). Yen-yong1 shared 20.00% of core genes with the closest relative, which was lower than the ICTV criteria for subfamily classification (27.00%‒79.00%). The results suggested that Yen-yong1 represented a novel genus and a novel subfamily belonging to Caudoviricetes. Yen-Yong1 was species-specific and capable of lysing 3 out of 4 tested Y. enterocolitica strains. Yen-yong1 maintained high bacteriolytic activity at -80‒60 ℃ and pH 2.0‒13.0, as well as in the presence of 0‒3% NaCl. Yen-yong1 can disrupt the biofilms of Y. enterocolitica. At 25 ℃ and MOI=100, the sterilization rates of Yen-yong1 against Y. enterocolitica CICC 10869, CICC 21669, and CMCC 52202 in raw pork slices were 93.48%, 52.77%, and 43.24%, respectively. All the 3 tested Y. enterocolitica strains proliferated at 4 ℃. At 4 ℃ and MOI=100, the sterilization rates of Yen-yong1 against Y. enterocolitica CICC 10869, CICC 21669, and CMCC 52202 in raw pork slices were 98.28%, 78.35%, and 96.33%, respectively. [Conclusion] Yen-Yong1 reveals a previously unknown evolutionary lineage. Yen-Yong1 has excellent biological characteristics and potential applications. This study enriches the knowledge of Y. enterocolitica phages, phage database, and gene database, laying a foundation for the application of phages in the prevention and control of food-borne yersiniosis.

[Objective] The enzymatic depolymerization of polyethylene terephthalate (PET) involves the hydrolysis of PET catalyzed by PET hydrolases, leading to the production of terephthalic acid and ethylene glycol. In this study, we identified a new PET hydrolase through gene mining, with the goal of providing enzymes for efficient depolymerization of waste PET. [Methods] A previously uncharacterized functional enzyme, named as AePETase, was identified through gene mining. This enzyme demonstrated the ability to hydrolyze amorphous PET films. Subsequently, heterologous expression, purification, and enzymatic characterization were performed for AePETase. [Results] AePETase showed a T_m value of (53.0±0.7) ℃, and an optimal reaction temperature of 45 ℃. The optimal buffer was Na₂HPO₄-NaH₂PO₄ (100 mmol/L, pH 8.0), and the optimal enzyme concentration was 1 µmol/L. After 72 h, the hydrolysis of amorphous PET films by AePETase resulted in 13.4-fold more products than that by the reported IsPETase. [Conclusion] AePETase demonstrates significant hydrolytic activity on PET and represents a promising new enzyme for hydrolyzing PET.

[Objective] To study the cytopathic effects (CPE) and replication characteristics of HCoV-OC43 VR1558 strain in cell lines derived from different species. [Methods] Thirteen cell lines from humans (MRC-5, HRT-18, Huh7, Huh7.5, RD, and HeLa), non-human primates (LLC-MK2 and Vero), and rodents (17Cl-1, Mv.1Lu, BHK-21, BHK-21-APN, and Neuro 2a) were selected and infected with HCoV-OC43 VR1558 strain. CPE were observed for several consecutive days. Virus-infected cells and supernatants were collected daily. RT-qPCR was conducted to monitor the changes in viral RNA copy number. The load of viruses with infectious ability was determined based on the tissue culture infectious dose 50% (TCID₅₀), and the kinetic curves of viral replication in different cell lines were established. [Results] Following infection with HCoV-OC43 VR1558 strain, CPE were observed in all the 13 cell lines. CPE primarily manifested as cell aggregation, shrinkage, rounding, and detachment. CPE appeared early in MRC-5, Mv.1Lu, HeLa, and 17Cl-1 cells, being noticeable within 72 h post-infection (hpi). The virus induced CPE in other cell lines after 120 hpi, and CPE were the mildest in HRT-18 and Huh7.5 cells. RT-qPCR results indicated that the viral RNA copy number increased most significantly within 24 hpi, although the time to reach the peak and the peak copy number varied among cell lines. Specifically, the RNA copy number in Huh7.5 cells reached the peak (10⁷ copies/mL) at 24 hpi, and that in 17Cl-1, BHK-21-APN, Mv.1Lu, and BHK-21 cells reached the peaks (10⁷ to 10⁹ copies/mL) at 48 hpi. In MRC-5, LLC-MK2, Neuro 2a, and Vero cells, the replication peaks (10⁶ to 10⁹ copies/mL) occurred at 72 hpi. In HRT-18, HeLa, Huh7, and RD cells, the viral RNA copy number peaked after 96 hpi, reaching 10⁸ to 10⁹ copies/mL. TCID₅₀ assay results demonstrated rapid viral proliferation within 24 hpi, while the time to reach the peak titer and the peak titers varied. The peak titer (2.68× 10⁷ TCID₅₀/mL) in BHK-21-APN cells was observed at 48 hpi. In BHK-21 and Neuro 2a cells, the peak titers (10⁶ to 10⁷ TCID₅₀/mL) were observed at 72 hpi. In MRC-5, 17Cl-1, HeLa, Huh7, Huh7.5, Mv.1Lu, LLC-MK2, and RD cells, the peak titers (10⁶ to 10⁸ TCID₅₀/mL) were observed at 96 hpi. In Vero cells, the virus strain reached the peak titer (10⁵ TCID₅₀/mL) at 120 hpi, while the strain reached the peak titer of 10⁸ TCID₅₀/mL in HRT-18 cells at 144 hpi. [Conclusion] HCoV-OC43 VR1558 strain exhibits a wide spectrum of cell tropism, demonstrating rapid replication and proliferation within 24 hpi across 13 cell lines derived from various species. However, the time to reach the replication peak varied among different cell lines. The highest viral titer achieved was 10⁸ TCID₅₀/mL, observed in MRC-5 and HRT-18 cells. This study provides experimental reference for further investigation of the replication characteristics, infection mechanism, and pathogenicity of HCoV-OC43, as well as for the screening and evaluation of antiviral drugs.

[Objective] To understand the molecular functions and potential applications of the significantly up-regulated gene cluster chr1_2605-chr1_2604 in response to tetrabromobisphenol A (TBBPA) stress, we investigated the roles of chr1_2605 and chr1_2604 in the specific recognition and efficient degradation of TBBPA. [Methods] Synthetic biology methods were employed to construct Sphingobiumxenophagum C1 (pBBR-2605-HiBiT) and Escherichiacoli BL21(DE3, pET30b-2604) as chassis cells for biosensing and degrading, respectively. The response characteristics of Chr1_2605 in the chassis cells to different pollutants were analyzed by the luciferase activity assay. Additionally, the degradation activity of TBBPA by Chr1_2604 in the chassis cells was determined by high-performance liquid chromatography. [Results] The xenobiotic-responsive element Chr1_2605 exhibited a highly specific response to TBBPA. The Chr1_2605-based chassis cell of S. xenophagum C1 (pBBR-2605-HiBiT) demonstrated high responsivity and sensitivity to TBBPA, with a limit of detection ranging from 0.010 to 0.050 μmol/L. The 2-oxoglutarate/Fe-dependent dioxygenase Chr1_2604 in the chassis cell of E. coli BL21(DE3, pET30b-2604) displayed the degradation rate of 44.415% for 2.0 mg/L TBBPA within 3 d [0.296 mg/(L·d)], which was significantly higher than those of most reported microbial strains under non-co-metabolic conditions. [Conclusion] The chr1_2605-chr1_2604 gene cluster can accurately recognize and degrade TBBPA. Specifically, the xenobiotic-responsive element Chr1_2605 specifically recognizes TBBPA, whereas the 2-oxoglutarate/Fe-dependent dioxygenase Chr1_2604 efficiently degrades TBBPA.

Lactic acid bacteria (LAB) are the predominant probiotics with significant health-promoting potential. Xizang pigs, an invaluable indigenous breed in China, harbor a unique and largely unexplored reservoir of intestinal LAB. [Objective] To screen high-quality LAB isolated from Xizang pigs. [Methods] LAB were isolated by the streak plate method and identified by 16S rRNA gene sequencing. Two Limosilactobacillus reuteri strains with significantly different lactic acid production capacities were selected from Xizang pigs. The two strains, together with one L. reuteri strain previously preserved in our laboratory, which originated from duroc×landrace×yorkshire pigs, were assessed in terms of acid and bile salt tolerance, antioxidant activity, antibacterial properties, and antibiotic resistance. [Results] A total of 21 LAB strains were isolated and identified from the intestinal microbiota of Xizang pigs, including ten Streptococcus alactolyticus strains, six L. reuteri strains, three Lactobacillus amylovorus strains, one Leuconostoc mesenteroides strain, and one Limosilactobacillus vaginalis strain. Among the three strains screened out, L. reuteri T-B5L2 exhibited the highest lactic acid production. Moreover, this strain demonstrated strong survival under pH 3.0 and 0.1% bile salts. L. reuteri T-B5L2 exhibited the strongest inhibitory activity against enteropathogenic Escherichia coli and Salmonella Choleraesuis. There were no significant differences in antioxidant activity among the three strains. All the three strains exhibited high sensitivity to penicillins and cephalosporins but displayed resistance to tetracyclines, aminoglycosides, and glycopeptides. [Conclusion] In this study, 21 LAB strains were isolated from the feces of Xizang pigs, among which L. reuteri T-B5L2 exhibited the highest acid production capacity. Further in vitro biochemical characterization demonstrated that this strain exhibited good growth performance, acid and bile salt tolerance, antioxidant activity, and the ability to inhibit pathogenic bacteria. These findings suggest that L. reuteri T-B5L2 is a promising probiotic candidate with potential applications in improving intestinal health and mitigating pathogenic infections.

[Objective] To clarify the diversity characteristics of gut microbiota in the important wild pollinator, Xylocopa appendiculata, and provide theoretical basis for the conservation and utilization of wild pollinator resources. [Methods] Utilizing employed 16S rRNA gene high-throughput sequencing, we conducted comparative analyses of gut microbiota diversity in adults X. appendiculata from different habitats and sexes. Functional prediction of gut microbial communities was performed using PICRUSt2. [Results] The gut microbiota in adults X. appendiculata was predominantly composed of Bacillota (relative abundance of 50.17%), Pseudomonadota (relative abundance of 32.04%), Actinomycetota (relative abundance of 12.84%), and Bacteroidota (relative abundance of 3.77%). The genus Lactobacillus was identified as a core microbiota present in all sampled individuals. Significant differences in the relative abundance of gut microbiota were observed across habitats and sexes. The samples from the Sui and Tang Dynasties City Ruins Botanical Garden habitat (HXST) exhibited the highest richness, diversity, and evenness of gut microbiota. In the Sui and Tang Dynasties City Ruins Botanical Garden habitat (ST) and the Caoyao Village habitat (CY), female adults showed higher microbial richness, diversity, and evenness compared to males. In the Shuiquan Village habitat (SQ), male adults showed higher microbial richness, diversity, and evenness compared to females. The genus Gilliamella, as the dominant genus of male samples from Caoyao Village (HXCYM), accounts for a very small proportion in other samples. Functional prediction PICRUSt2 showed that the metabolic gene functions of the gut microbiota of adults X. appendiculata are the most enriched, with an abundance ratio of 74.00%. [Conclusion] The gut microbiota of adults X. appendiculata exhibits ecological adaptive features with social bees (e.g., Apis mellifera and Bombus spp.), characterized by low diversity, highly conservation, and specialized core microbial community structure. Environmental heterogeneity and host sex were identified as key drivers of gut microbiota divergence. These findings suggest that the gut microbiota of X. appendiculata may play a central role in maintaining host energy homeostasis and population adaptability.

[Objective] To explore the autophagy induced by the African swine fever virus (ASFV) protein E423R. [Methods] The RFP-LC3 fluorescent spots following the transfection of HeLa-Difluo^TM hLC3 cells with pCMV-Myc-E423R was quantified by the High-Content Analysis System, and the autophagic flux was assessed. The expression levels of key autophagy proteins, LC3-Ⅱ and SQSTM1/p62, in HeLa cells were analyzed via Western blotting. Co-localization of autophagosomes and lysosomes was examined by laser confocal microscopy. Additionally, Western blotting was employed to investigate the dose-dependent effect of E423R on LC3-Ⅱ expression and the regulatory effect of E423R on the AKT/mTOR/ULK1 signaling pathway. [Results] The High-Content Analysis System demonstrated a significant increase in RFP-LC3 fluorescence spots in the reporter cells expressing E423R, suggesting that E423R induced the activation of autophagy. Western blotting further confirmed that the expression of E423R significantly elevated the LC3-Ⅱ/β-actin ratio while decreasing the expression level of p62. Confocal microscopy results indicated that E423R enhanced the expression of GFP-LC3, promoted the co-localization of GFP-LC3 with Lyso-Tracker Red, and facilitated the fusion of autophagosomes and lysosomes. Additionally, the expression level of E423R exhibited a positive correlation with the expression level of LC3-Ⅱ in HeLa cells. Furthermore, E423R down-regulated the expression of p-AKT, p-mTOR, and p-ULK1 (Ser757) in the mTOR signaling pathway. [Conclusion] This study demonstrates that the ASFV E423R protein induces complete autophagy via the AKT/mTOR/ULK1 signaling pathway, exhibiting a dose-dependent effect on LC3-Ⅱ expression to a certain degree. These findings provide a foundation for further investigation into the infection and pathogenic mechanisms of ASFV.

[Objective] To improve the fermentation performance by optimizing the fermentation medium and conditions of Bacillus subtilis subsp. spizizenii YC25, thereby providing a theoretical basis for the subsequent industrial development and application of the strain. [Methods] With the spore yield as the indicator, the optimal fermentation conditions were screened by single factor tests. On this basis, the response surface methodology was employed to optimize the fermentation medium. Pot experiments were conducted to evaluate the control effects of the optimized fermentation broth on tobacco black shank and tobacco Fusarium root rot. [Results] The optimum fermentation conditions were as follows: 30 ℃, 180 r/min, an inoculation amount of 2%, a liquid-loading volume of 20%, and fermentation time 36 h. The optimum fermentation medium was as follows: corn starch 15.53 g/L, soybean meal 8.70 g/L, KH₂PO₄ 0.50 g/L, MgSO₄ 0.92 g/L, and CaCO₃ 0.50 g/L. Under these conditions, the spore yield reached 1.423 3×10¹⁰ CFU/mL. The results of pot experiments showed that the control effects of the fermentation broth diluted by 20 times on tobacco black shank and tobacco Fusarium root rot were 77.88% and 62.09%, respectively. [Conclusion] The optimized medium could significantly increase the spore yield of strain YC25, and the disease indexes of tobacco black shank and tobacco Fusarium root rot were significantly reduced after the application of YC25 fermentation broth. This study provides a theoretical basis for the development and application of the biocontrol strain YC25 and the prevention and control of field diseases in the future.

[Objective] To develop a new and rapid fluorescent recombinase-aided amplification (RAA) method for the detection of Enterocytozoon hepatopenaei (EHP), one of the major pathogens restricting the development of the prawn farming industry. [Methods] Multiple pairs of RAA primers were designed based on the highly conserved 18S rRNA gene of EHP, and the primers with the highest amplification efficiency were screened by basic RAA and fluorescent RAA. The reaction system and conditions of fluorescent RAA were then optimized. The optimization results showed that the developed method achieved detection at 37 ℃ within 20 min. Finally, the sensitivity, specificity, precision, stability, and actual sample application of the developed fluorescent RAA method were evaluated. [Results] The fluorescent RAA method had high specificity and was only sensitive to EHP, and it had no response to other pathogens causing mixed infection or secondary infection. Moreover, it had high sensitivity and high precision, with the limit of detection being 5 copies/μL and the coefficient of variation less than 5% for repeat test results. The lyophilized premix of primers and probe demonstrated good stability. It can be stably transported, used, and stored for 10 d at room temperature (25 °C) and 5 d at the high temperature (37 °C) in summer, with the shelf life longer than one year at -20 ℃. Additionally, the detection results of ninety clinical samples of prawn by the developed method were consistent with those obtained by the fluorescence quantitative PCR method recommended by fishery industry standard Code of Diagnosis for Enterocytozoon hepatopenaei Disease (SC/T 7232—2020), with the result coincidence rate of 97.78% (88/90). In addition, the fluorescent RAA method showed the sensitivity of 100.00% (30/30) and the specificity of 96.67% (58/60). [Conclusion] The established fluorescent RAA method has the characteristics of rapid detection, simple operation, high sensitivity, high specificity, high precision, and good stability, providing a technical reference for the field rapid detection of EHP. It has a wide market application prospect in the accurate and rapid detection of aquatic diseases.