Abstract:Listeria monocytogenes, a major zoonotic food-borne intracellular pathogen, is ubiquitous in the natural environment and easily contaminates animal-derived food products. The consumption of the contaminated food can cause severe listeriosis in both humans and animals, with the mortality rate reaching up to 30%. The antimicrobial therapy is the only feasible approach for treating L. monocytogenes infection since L. monocytogenes is susceptible to multiple antimicrobials. However, the reports of multidrug-resistant strains are increasing due to the selective pressure exerted by the irrational use of antimicrobials or disinfectants. The antimicrobial resistance mechanisms of L. monocytogenes are complex. Efflux pump proteins are crucial in bacteria and participate in various biological processes. Specifically, they can influence bacterial sensitivity to antibiotics, facilitate the efflux of toxic compounds, and affect bacterial virulence. Over the last two decades, scholars have conducted research on the efflux pumps-mediated resistance of L. monocytogenes, identifying several efflux pump proteins associated with the efflux of antibiotics or toxic compounds. Additionally, some efflux pumps are involved in the virulence expression process of L. monocytogenes. This paper reviews the research advances in the functions and regulatory mechanisms of efflux pumps in multidrug-resistant L. monocytogenes. It provides a theoretical foundation for probing into the environmental adaption mechanisms of L. monocytogenes, curbing the spread of this pathogen, and identifying new drug targets for combating infections.

Abstract:Persulfidation plays a role in protein functioning and signaling, maintaining the physiological and metabolic balance of cells, protecting cells from oxidative stress, and regulating sulfur homeostasis. This article summarized the internal relationship of hydrogen sulfide, reactive sulfur species, and cysteine metabolism, expounded the mechanism of sulfur homeostasis regulation, and introduced the role of persulfidation in microbial sulfur homeostasis, providing new thoughts for the future research.

Abstract:The roles of gut microbiota and its metabolites in the pathogenesis and rehabilitation of neurodegenerative diseases, gastrointestinal diseases, and musculoskeletal systemic diseases in the elderly are receiving increasing attention. Gut microbiota and its metabolites can regulate the functions of the cranial nervous system and the musculoskeletal system through various pathways, involving the immune, endocrine, and nervous systems. Conversely, the gut, brain, and musculoskeletal system can act on the intestinal system via inflammatory, metabolic, and mitochondrial pathways to regulate the gut microbiota. Accordingly, bidirectional signaling mechanisms are formed via the gut-brain, gut-muscle, and gut-brain-muscle axes, which affect the organism health. This review summarizes that gut microbiota establishes gut-brain-muscle interconnections mainly through metabolites, intestinal permeability, and immune-neural pathways, providing new ideas for improving the brain neuroplasticity and muscle health.

Abstract:Atopic dermatitis (AD) is a highly prevalent allergic skin disease characterised by recurrent attacks and severe itching. The pathogenesis of AD involves a variety of factors including genetic susceptibility, epidermal barrier dysfunction, microbiome dysbiosis, immune imbalance, and the environment, while the available therapeutic drugs have severe side effects and limited efficacy. Studies have demonstrated that gut microbiota, particularly probiotics, play a role in AD. Probiotics can alleviate AD symptoms by inhibiting pathogens, enhancing barriers, improving the intestinal environment, and balancing the Th1/Th2 immune response, among other mechanisms. In this review, we summarized the skin and intestinal microecological characteristics of AD patients and systematically elucidated the mechanisms of probiotics in alleviating AD from the pathogenesis and influencing factors of AD, aiming to provide theoretical support for probiotics in the treatment of AD and related allergic skin diseases.

Abstract:Hepatitis E virus (HEV) is a major zoonotic pathogen that causes acute viral hepatitis worldwide. HEV has high genetic diversity, and the incidence of various genotypes or subtypes is strongly correlated with host species, geographic location, and prevention and control methods. HEV strains HEV-3, comprising 3a-3i subtypes, were present in Europe and America, whereas HEV-3 and HEV-4 were prevalent in Asia. The epidemic strains in China have evolved from HEV-1 to HEV-4. Recent studies have shown that gene recombination, amino acid mutations, and synonymous codon usage patterns are part of the mechanisms underlying HEV evolution. In particular, amino acid mutations are the main driving force for the continued prevalence of the virus. This paper reviews the classification, global epidemic characteristics, and evolutionary mechanism of HEV, aiming to provide a reference for the prevention and control of hepatitis E and vaccine development.

Abstract:Adequate intake of probiotics helps in maintaining the homeostasis of the gut microbiota, which is of great significance for human intestinal health. However, probiotics are vulnerable to stress and necessitate stringent storage conditions in industrial settings, which pose challenges to the vitality maintenance of probiotics in transportation and shelf-life. Therefore, it is essential to develop the preparation process capable of protecting the high vitality of probiotics. Drying beneficial for sustaining probiotic vitality is often used to maintain the strain stability. Among the drying methods, freeze-drying is widely used. However, during the freeze-drying process, probiotics encounter environmental stress conditions, which lead to cellular damage and even death. Therefore, the freeze-drying protectants that can significantly improve probiotic survival rate have become a research hotspot in the industrial application of probiotics. This article systematically summarizes both the commonly used and the newly discovered protectants for freeze-drying of probiotics and elucidates their mechanisms of action. It provides an overview of the factors affecting cell viability post freeze-drying while outlining the prospective research directions of freeze-drying protectants. This review is expected to furnish theoretical substantiation for the development of freeze-dried probiotics with high vitality.

Abstract:[Objective] This study aims to investigate the soil fungal community structure in the tea plantations of Heshan and explore the relationship between fungal community and soil physicochemical properties. [Methods] We employed high-throughput sequencing to analyze the soil fungal community composition of 49 rhizosphere soil samples collected from healthy and mismanaging tea plantations in Heshan. The redundancy analysis (RDA) was performed to analyze the effects of soil physicochemical properties on the fungal community structure. The pairwise Wilcoxon signed-rank test was performed to compare the fungal taxa between the two kinds of tea plantations. FUNGuild was used to predict the soil fungal functions. [Results] Basidiomycota, Mortierellomycota, and Ascomycota were the three dominant phyla in the rhizosphere soil of tea plantations in Heshan. The mismanagement of tea plantations increased the relative abundance of Basidiomycota, but significantly decreased the relative abundance of Mortierellomycota. The fungal richness, Chao1 index, and ACE index were significantly lower in the mismanaging tea plantation than in the healthy tea plantation. The total nitrogen, total phosphorus, available phosphorus, organic matter, and available nitrogen were the primary driving factors for the differences of soil fungal β diversity. The soil pH, total phosphorus, exchangeable Ca²⁺ and Mg²⁺, available phosphorus, and available potassium were significantly correlated with the fungal taxa. Compared with the healthy tea plantation, the mismanaging tea plantation showed decreased relative abundance of core fungal taxa but increased relative abundance of medium and rare taxa. Ten of OTUs was common between healthy and mismanaging tea plantations. The pathogens causing tea gray blight, i.e., Pseudopestalotiopsis theae and Fusarium keratoplasticum, were identified in the healthy tea plantation. The relative abundance of biocontrol fungi, Trichoderma spirale and T. atroviride, significantly increased in the mismanaging tea plantation. The mismanaging tea plantation demonstrated increased relative abundance of pathotrophic, pathotrophic-saprotrophic-symbiotrophic, and symbiotrophic fungi but decreased relative abundance of saprotrophic-symbiotrophic fungi. [Conclusion] We elucidated the relationship of management mode with fungal community composition and soil physicochemical properties, providing the insights into the prevention and control of pathogens infecting black tea and the screening of biocontrol fungi in Heshan.

Abstract:[Objective] Streptococcus thermophilus is widely used in the dairy industry as a common starter for fermented dairy products such as yogurt and cheese. Most strains of S. thermophilus are galactose-negative (Gal^-) and unable to metabolize galactose and excrete it extracellularly, which results in an increase in the galactose content in fermented milk. S. thermophilus can be treated by chemical mutagenesis to metabolize galactose and then used to develop the fermented milk products with low galactose content. [Methods] We used nitrosoguanidine (NTG) to induce the mutation of S. thermophilus IMAU80846. Furthermore, we measured the activities of β-galactosidase (β-Gal), galactokinase (GalK), pyruvate kinase (PK), and glucokinase (GK) in the wild-type and mutant strains of S. thermophilus IMAU80846 and analyzed the amino acid sequences encoding these enzymes. S. thermophilus IMAU80846Y that could metabolize galactose was obtained. We performed whole genome sequencing of the mutant strain and measured the content of lactic acid, lactose, galactose, and glucose in the fermented milk products produced with the wild-type strain and the mutant strain. We then compounded the wild-type strain and the mutant strain with Lactobacillus delbrueckii subsp. bulgaricus IMAU20450, respectively, and characterized the two groups of fermented milk products during fermentation and storage. Finally, we prepared a fermented milk product with low galactose content. [Results] S. thermophiles IMAU80846Y had higher activities of β-Gal and GalK and lower activities of PK and GK than the wild-type strain. The amino acid sequences and whole genome sequences showed that the mutant strain had mutations in the genes involved in carbohydrate metabolism. The HPLC results showed that the fermented milk produced with the mutant strain had lower content of lactose and galactose and higher content of lactic acid and glucose than that produced with the wild-type strain. Compared with the compound group with the wild-type strain, the compound group with the mutant strain improved the titration acidity, viable cell count, viscosity, and water holding capacity of fermented milk. [Conclusion] The mutagenesis with NTG changed the ability of S. thermophilus IMAU80846 to metabolize galactose, and the mutant strain could be used to produce the fermented milk with low galactose content.

Abstract:[Objective] The biofloc-based culture system (BFS) of Penaeus vannamei is a new ecological shrimp production mode based on the concept of cultivating and regulating the microbial community in the aquaculture system. However, the characteristics and assembly processes of microbial communities in different habitats of the BFS remain unclear. [Methods] The 16S rRNA gene sequencing was employed to explore the bacterial community composition in three different habitats (water, bioflocs, and shrimp gut) of the BFS. SourceTracker and the neutral model were adopted to explore the characteristics and assembly processes of bacterial communities in different habitats. [Results] The bacterial community diversity and composition varied significantly in the three habitats, and the structures and composition of bacterial communities were similar between bioflocs and shrimp gut. SourceTracker results showed that 98.76% of bacterial taxa in the shrimp gut were sourced from bioflocs, and only 0.83% were derived from water. Ruegeria existed in all the three habitats, with the relative abundance of 1.72%, 7.34%, and 6.00% in water, bioflocs, and shrimp gut, respectively. The unique amplicon sequence variants (ASVs) showed the maximal number of 89 in water, mainly belonging to Mariculis and Owenweeksia. Bioflocs contained 56 ASVs, which were mainly Rheinheimera. Only 10 ASVs were present in the shrimp gut, mainly belonging to Roseobacter. The bacterial communities in water, bioflocs, and shrimp gut were all fit to the neutral model, which indicated that the bacterial communities in the three habitats were dominated by the neutral process. [Conclusion] The microbial communities in different habitats of the BFS were significantly different, and the intestinal bacteria of shrimp mainly came from bioflocs. The assembly processes in the three habitats were dominated by neutral processes. The results provide a theoretical basis for regulating the microbial community in the BFS.

Abstract:Pathogenic mycoplasma has the ability to invade host cells, which is a key mechanism to their pathogenicity. Functional proteins that mediate the entry of mycoplasma may serve as potential drug or vaccine targets. [Objective] To clone and express the LRR5 protein encoded by MBOVPG45_0564 in Mycoplasma bovis and to explore its role in the invasion of host cells by M. bovis. [Methods] The NCBI database was used for the homology analysis of MBOVPG45_0564, and the structure of LRR5 protein was predicted by the Discovery Studio Client system. After prokaryotic expression of LRR5 protein, the mouse polyclonal antibody was prepared, and the subcellular localization of LRR5 protein was observed by immunoelectron microscopy. The invasion of M. bovis into embryonic bovine lung (EBL) cells after LRR5 antibody blocking was observed by plate counting and laser confocal microscopy. After conjugation of LRR5 protein to the surface of fluorescent microspheres, the entry of the microspheres into EBL cells was observed by laser confocal microscopy and a high-content live-cell imaging system. [Results] MBOVPG45_0564 was a conserved sequence in M. bovis and encoded LRR5, a membrane-associated protein with a typical crescent-like spatial conformation. Multiple repeating leucine motifs were assembled in a supercoiled manner to form a solenoid protein structural unit. After LRR5 antibody blocking, the invasion rate of M. bovis to EBL cells reduced (P<0.05), and the fluorescent microspheres conjugated with LRR5 protein could successfully enter EBL cells. [Conclusion] The LRR5 protein encoded by MBOVPG45_0564 is localized on the M. bovis membrane and plays a role in the invasion of M. bovis into host cells.

Abstract:Equine coronavirus (ECoV) is an emerging virus attacking the gastrointestinal tract in horses, and the infected adult horses mainly present fever, abdominal pain, and diarrhea. In 1975, ECoV infection first appeared in the United States, and since then it has been prevalent in many countries and regions. Only one recombinant strain of ECoV was isolated from the small intestine of a donkey experiencing diarrhea in Shandong Province, China. [Objective] Understanding the genetic composition, genetic relationship, and biological characteristics of ECoV strains in China can lay a foundation for unveiling the epidemic status and genetic evolution trend of ECoV and provide materials for the research and development of products for the prevention and control of ECoV. [Methods] RT-PCR was employed to detect the stool samples from a horse experiencing diarrhea in Huangpi District, Wuhan City, Hubei Province. The virus was isolated from the positive samples and verified by indirect immunofluorescence assay (IFA) with monoclonal antibodies targeting the S1 protein of ECoV. Based on the whole genome sequencing results of the isolate, the phylogenetic analysis and sequence alignments of the whole genome, N gene, and NS2 gene were performed. [Results] An ECoV strain was successfully isolated and named ECoV-JL. Transmission electron microscopy (TEM) showed that the isolated virus particles were spheroidal and had a capsule membrane and a typical spiroid structure of coronaviruses. The tissue culture infectious dose 50% (TCID₅₀) of ECoV-JL reached a peak of 10^6.16 TCID₅₀/mL 72 h post infection in HRT-18 cells. ECoV-JL strains could infect three human cell lines: HRT-18 (human ileocecal cancer cells), Caco-2 (human colorectal adenocarcinoma cells), and Huh7 (human liver cancer cells). The genome sequence of ECoV-JL and the ECoV genome sequences in GenBank showed the similarity within the range of 97.9%–99.0%. ECoV-JL was in a separate branch of the phylogenetic tree and far related to other strains, which indicated that ECoV-JL might be derived from recombination mutations. The NS2 gene presented more mutations, and the difference in NS2 gene was the main reason for the poor homology between ECoV-JL and other strains. [Conclusion] We isolated and identified an ECoV strain from the stool samples of horses with diarrhea and named it ECOV-JL. The study about the biological characteristics and phylogenetic relationship of this strain reflected the characteristics of the epidemic strains in Hubei Province, providing a clue for the epidemic status and evolution trend of ECoV in China.

Abstract:[Objective] Prolidase is an enzyme that can hydrolyze proline or hydroxyproline residues from the C-terminal dipeptides (Xaa-Pro). A putative prolidase-encoding gene was identified in the genome of Pyrococcus yayanosii CH1 isolated from the deep sea. In this study, we characterized the enzymatic properties of Pyprol encoded by PYCH_07700 in vitro, aiming to find a new prolidase. [Methods] Pyprol was heterologously expressed in the hyperthermophilic archaeon Thermococcus kodakarensis TS559. The dipeptide Met-Pro was used as a substrate to test the prolidase activity of the purified recombinant protein. [Results] Pyprol showed the best performance at 100 °C and pH 6.0. Pyprol binding to Co²⁺ exhibited the maximum activity, and the optimal metal ion concentration was 1.2 mmol/L. Pyprol had catalytic activity in a wider pH range and can tolerate higher concentrations of metal ions than the prolidase Pfprol from P. furiosus. Pyprol was a piezotolerant protein with an optimal hydrostatic pressure of 40 MPa. It exhibited enhanced activities at 40, 70, and 100 °C under 40 MPa, compared with at the atmospheric pressure. [Conclusion] Pyprol is a novel thermostable and piezotolerant prolidase of P. yayanosii CH1, which is an obligate piezophilic hyperthermophilic archaeon strain isolated from a deep-sea hydrothermal vent.

Abstract:[Objective] Listeria monocytogenes (Lm) is a ubiquitous foodborne pathogen causing listeriosis. Lm can grow at low temperatures and thus may cause safety problems of refrigerated food and threaten the public health. The growth of Lm at low temperatures involves the inhibition of flagellar gene expression, which restricts flagellar biosynthesis. MogR is a transcriptional repressor which represses the expression of flagellar genes during intracellular infection and during extracellular growth of Lm at 37 ℃, resulting in no biosynthesis of flagella. Whereas MogR is deprived of repression function and the bacteria produce flagella during growth at 20–30 ℃. Our studies demonstrated that Lm significantly reduced the flagellar production at 4 ℃, but the molecular mechanism of which remained unclear. This study aims to reveal the relationship between the reduction of flagella and MogR repression at 4 ℃. [Methods] We constructed the mogR-deleted mutant ΔmogR and the flagellin gene flaA-deleted mutant ΔflaA (as the control strain with no flagella), and their complementary strains cΔmogR and cΔflaA with the Lm strain ATCC 19115 as the parental strain. Then, we analyzed the swarming motility, flagellar biosynthesis, and transcriptional levels of flagellar genes in above five strains at 4 ℃, 28 ℃, and 37 ℃, respectively. The growth curves of these strains were determined at 4 ℃, 28 ℃, and 37 ℃, respectively. [Results] Compared with the parental strain, ΔmogR showed significantly increased in motility, flagellar biosynthesis, and transcriptional levels of flagellar genes (P<0.01, 0.001, and 0.001, respectively) at 4 ℃. The growth of ΔmogR markedly decreased compared with the parental strain (P<0.05) at 4 ℃. The data of motility, flagellar biosynthesis, and transcriptional levels of flagellar genes in cΔmogR had no significant differences compared with the parental strain. [Conclusion] The reduction in flagellar biosynthesis was associated with the repression function of MogR in Lm at 4 ℃. The reduction in flagellar biosynthesis was of benefit to Lm proliferation at low temperatures. This study enriched our understanding of the mechanism of Lm growth at low temperatures.

Abstract:[Objective] To identify efficient aerobic denitrifying bacteria in the phyllosphere and provide new ideas for the prevention and control of air pollution by phytoremediation. [Methods] We used the enrichment culture method combined with the Griess reagent and bromothymol blue to isolate aerobic denitrifying bacteria from the phyllosphere and analyzed the phylogenetic relationship of the isolates based on the 16S rRNA gene sequences. Efficient aerobic denitrifying bacteria were screened out and their denitrification conditions were optimized. [Results] Thirteen strains were isolated from the phyllosphere of six landscape plant species: Photinia serratifolia, Ligustrum lucidum, Osmanthus fragrans, Camphora officinarum, Euonymus japonicus, and Magnolia grandiflora. According to the 16S rRNA gene sequences, the 13 strains were classified into 7 genera, 7 families of 4 phyla. Specifically, 4, 3, and 2 strains belonged to Enterobacter, Achromobacter, and Pseudomonas, respectively, and the remaining 4 strains belonged to Acinetobacter, Sphingobacterium, Microbacterium, and Pseudarthrobacter. The comparative analysis revealed that strain SF outperformed the other strains in denitrification. The factors (carbon source, temperature, initial pH, C/N ratio, and rotational speed) influencing the denitrification performance of SF were optimized by single factor tests and response surface methodology. The denitrification conditions of SF were optimized as follows: glucose as the carbon source, initial pH 7.5. C/N ratio of 9.7, rotational at 180 r/min, and temperature of 33.5 ℃. Under the optimized conditions, the total nitrogen removal rate reached 93.3% in 72 h when the initial nitrate concentration was 361 mg/L. [Conclusion] The rich culturable aerobic denitrifying bacteria in the phyllosphere of landscape plants laid a foundation for phytoremediation. It is feasible to synergistically reduce nitrogen oxide pollution in the air by screening efficient denitrifying strains and improve the denitrifying performance by optimizing the culture conditions.

Abstract:Heyndrickxia coagulans characterized by low nutrient requirements, high titers of fermentation products, and thermal tolerance has become a major microbial species for lactic acid fermentation. We have demonstrated that phosphate stimulates the gene expression of l-lactate dehydrogenase in H. coagulans to increase l-lactic acid production, the mechanism of which, however, remains unknown. [Objective] To primarily investigated the mechanism of phosphate in stimulating the gene transcription of lactate dehydrogenase in H. coagulans. [Methods] RT-PCR was employed to analyze the transcriptional level changes of lactate dehydrogenase in H. coagulans after phosphate addition. The core promotor region responsive to phosphate stimulation was identified by conventional methods of molecular biology. [Results] The key element responsive to phosphate was located in the upstream promoter region of the l-lactate dehydrogenase gene. The core region of the promoter was responsible for phosphate stimulation. The identified promoter was employed to promote d-lactate production with diammonium phosphate addition. [Conclusion] This study reported a new phosphate-responsive gene element, providing a theoretical basis for improving the synthesis efficiency of other biochemicals.

Abstract:[Objective] We investigated the diversity, composition, and functions of soil bacterial communities of wild Glycyrrhiza uralensis Fisch., a typical halophyte and medicinal plant, in the habitats with different degrees of salinization. The study can help to reveal the linkage between soil salinity and microbiome related to the growth, development, and quality formation of G. uralensis, being essential for improving the quality of cultivated G. uralensis. [Methods] Soil samples were collected from six main habitats of wild G. uralensis. High-throughput sequencing was employed to compare the diversity, composition, and functions of soil bacterial communities among the habitats of un-salinization (US), light salinization (LS), moderate salinization (MS), and heavy salinization (HS) and to excavate the dominant bacteria in different habitats. [Results] The soil bacterial richness and diversity of wild G. uralensis were higher in LS and MS habitats than in US and HS habitats and were the lowest in the HS group. The principal component analysis (PCA) revealed differences in the composition and functions of soil bacterial communities between groups with different degrees of salinization (P<0.05). The redundancy analysis (RDA) showed that total salinity (TS) was an important factor influencing the composition and functions of the soil bacterial community in the native habitat. The dominant bacterial genera in the US and LS groups were the same, all of which were plant-beneficial bacteria, including Nocardioides, Streptomyces, and Marmoricola. The significantly dominant genera in the MS group included both the beneficial bacteria unidentified_Acidobacteria and the halophilic bacteria Halomonas and Marinobacter. The soil bacteria in the HS group were dominated by salinophilic or salinity-tolerant bacteria, including Halomonas, Marinobacter, Truepera, Alifodinibius, Salinimicrobium, and Salegentibacter. The PICRUSt prediction results underlined the potential of soil bacteria in interactions with plants in US, LS, and MS groups, suggesting that beneficial bacteria in the three habitats influenced the growth, development, and quality formation of wild G. uralensis. Moreover, the prediction results emphasized that the soil bacteria endowed the HS group with the potential of self-repairing for adaptation to highly saline environments and improving the salt tolerance of wild G. uralensis. This result suggested that halophilic and salt-tolerant bacteria in the HS habitat played a key role in the salt tolerance of wild G. uralensis. The MS habitat had both kinds of bacteria, being worthy of attention. [Conclusion] The soil bacterial diversity and richness of wild G. uralensis were remarkably higher in LS and MS habitats than in US and HS habitats. The composition and functions of soil bacterial communities in US and LS habitats were similar and differed significantly from those of the HS habitat, and the MS habitat had characteristics of both the above.

Abstract:The conventional rescue system of newcastle disease virus (NDV) contains a cDNA clone plasmid and three helper plasmids expressing NDV: nucleocapsid protein (NP), phosphoprotein (P) and polymerase protein (L), respectively. These four plasmids have to be transfected into the same host cell simultaneously to complete the assembly of the virus, which is relatively inefficient. [Objective] To improve the rescue efficiency of NDV, this study aims to establish a two-plasmid rescue system. [Methods] The expression cassettes of NP, P, and L genes were constructed and sequentially cloned into the eukaryotic expression vector pCI to generate a single-helper plasmid, named pCI-NPL, capable of co-expressing NP, P, and L proteins. During this process, the genomic cDNA of the NDV LaSota strain was incorporated downstream of the CMV promoter in the pCI expression vector. Simultaneously, the reporter gene EGFP was inserted into the genome between the P and M genes, accompanied by the introduction of a hammerhead ribozyme sequence at the 5′ end and a hepatitis delta virus ribozyme sequence at the 3′ end. This culminated in the development of the full-genome transcriptional plasmid termed pCI-LaSota-EGFP. The two plasmids pCI-LaSota-EGFP and pCI-NPL were co-transfected as a two-plasmid system into BHK-21 cells to rescue the recombinant virus rLaSota-EGFP. The biological characteristics of the virus were then examined. [Results] RT-PCR, fluorescence microscopy, Western blotting, and growth characterization confirmed that rLaSota-EGFP was correctly constructed and expressed the foreign gene. The rescued recombinant virus rLaSota-EGFP had similar biological characteristics to wild-type (WT) LaSota. [Conclusion] A novel two-plasmid rescue system for NDV based on the CMV promoter was successfully established, laying a foundation for the efficient rescue of recombinant NDV and other paramyxoviruses.

Abstract:Fusarium head blight (FHB) caused by Fusarium graminearum is one of the major diseases limiting wheat production. Biocontrol has been considered an effective and sustainable method for the control of crop diseases. [Objective] To screen out endophytic strains with inhibitory effects on F. graminearum from wheat grains and evaluate their biocontrol potential, providing strain resources and theoretical support for the development and utilization of biocontrol agents against FHB in wheat. [Methods] Plate confrontation, spore germination, and cell-free supernatant (CFS) inhibition experiments were carried out to screen out the endophytic strains with antagonistic activity against F. graminearum from wheat grains. Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) were employed to observe the conidial morphology, membrane integrity, and mycelial reactive oxygen species of F. graminearum treated with the CFS. Pot experiments were performed to verify the biocontrol effects of the endophytic bacteria on FHB. Illumina HiSeq was used for whole genome sequencing. [Results] A highly efficient endophytic strain JB7 inhibiting the growth of F. graminearum was isolated from wheat grains. The CFS of strain JB7 in the decline stage showed the inhibition rate of 85.23% on the spore germination of F. graminearum. Moreover, it led to concavity on spore surface, cell membrane damage, leakage of nucleic acids and proteins, and accumulation of reactive oxygen species in the mycelia of F. graminearum. The CFS of strain JB7 significantly increased the content of soluble protein and malondialdehyde. Strain JB7 could produce protease, cellulase, glucanase, and siderophores. Pot experiments showed that strain JB7 decreased the disease index of FHB (P<0.05) in wheat. The strain was identified as Bacillus methylotrophic JB7 by whole genome sequencing, and it carried 12 gene clusters for the synthesis of secondary metabolites with antimicrobial functions. [Conclusion] Strain JB7 could inhibit the growth of F. graminearum and demonstrated a strong control effect on FHB, serving as a candidate strain for the biocontrol of FHB in wheat.

Abstract:[Objective] To analyze the polysaccharide hydrolysis activity and genomic characteristics of a Gram-negative bacterial strain FZY0027 isolated from intertidal seawater. [Methods] The strain FZY0027 was identified based on the morphological characteristics, 16S rRNA gene sequence, and the whole genome sequence determined by Illumina NovaSeq and Oxford Nanopore PromethION. Bioinformatics tools such as dbCAN, EasyCGTree, BRIG, and Easyfig were used to compare the strain FZY0027 with Saccharophagus degradans 2-40^T. The 3,5-dinitrosalicylic acid (DNS) method was employed to measure the polysaccharide hydrolysis activity of strain FZY0027. [Results] The 16S rRNA gene sequence showed the similarity of 99.9% between strain FZY0027 and S. degradans 2-40^T, and thus strain FZY0027 was preliminarily identified as S. degradans FZY0027. The highest levels of reducing sugars (2.28, 1.75, and 1.10 mg/mL, respectively) were produced by FZY0027 through the hydrolysis of starch, xylan, and mannose. The genome of strain FZY0027 was 5 178 381 bp, encoding a total of 4 156 genes, with the G+C content of 45.8%. The average nucleotide identity (ANI), average amino acid identity (AAI), and digital DNA-DNA hybridization (dDDH) values between strain FZY0027 and S. degradans 2-40^T were 96.5%, 96.7%, and 70.0%, respectively. A total of 303 genes were annotated in the Carbohydrate-Active Enzyme database, and there was a significant difference in the number (137 and 130, respectively) of genes encoding glycoside hydrolases (GHs) between strain FZY0027 and S. degradans 2-40^T. Strain FZY0027 carried multiple genes involved in the hydrolysis of starch and xylan, which was corresponding to its strong ability to hydrolyse starch and xylan. However, compared with S. degradans 2-40^T, strain FZY0027 could only hydrolyse a few polysaccharides under the experimental conditions in this study, which suggested that this strain may require specific culture conditions to fully exert its polysaccharide hydrolysis ability. [Conclusion] Strain FZY0027 is a versatile polysaccharide-hydrolyzing bacterium with the potential for bioresource utilization.

Abstract:[Objective] The microecological effects of transgenic plants with Bt and Bar genes are an important aspect of environmental safety assessment. However, few studies concern the impacts of rice genotypic alterations induced by Bt and Bar transformation on the microbial community composition and potential functions in different tissues of rice plants. [Methods] High-throughput sequencing of bacterial 16S rRNA gene and fungal ITS was performed to analyze the microbial community structure and potential functions in the rhizosphere soil, roots, stems, and leaves of Bt and Bar transgenic rice T1C-1 and its parent Minghui63 (control) at the heading stage. [Results] The bacterial and fungal diversity varied among different tissues in rice plants, being significantly higher in the underground niches (rhizosphere soil and roots) than in the aboveground parts (leaves and stems). T1C-1 significantly affected the Shannon index and Simpson index of endophytic fungi in leaves but had no significant effect on the microbial diversity in the stems, roots, or rhizosphere soil. The endophytic fungi Aspergillus and Talaromyces showed increased relative abundance in the leaves of T1C-1, which suggested their involvement in processes such as carbon metabolism, energy metabolism, and transcription. The average clustering coefficient and average degree of the microbial communities in T1C-1 were significantly higher than those in Minghui63, indicating that T1C-1 increased the complexity of the microbial community network. Phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt2) was employed to predict the functional enzyme genes of endophytic fungi in the leaves, which showed that T1C-1 significantly altered the pathways such as carbon metabolism, lipid metabolism, and energy metabolism compared with Minghui63. [Conclusion] The community composition and potential functions of endophytic fungi in leaves were more sensitive to T1C-1 than those in the rhizosphere soil, while T1C-1 did not decrease the diversity of endophytic fungi in leaves. More attention should be paid to the diversity changes of endophytic microorganisms in different ecological niches of plant tissues in the evaluation of the microecological effects of transgenic plants.

Abstract:[Objective] To investigate the diversity of bacteria and fungi in the pathosphere of Plasmopara viticola and screen out the strains with potential biocontrol effects on grape downy mildew. [Methods] The leaves infected by P. viticola were collected from seven representative grape-producing regions in northern and southern China in two consecutive years. The collected leaves were cultured in a humid environment, and the newly growing downy mildew was aseptically picked by forceps to prepare the sporangial suspensions of P. viticola. The strains were isolated by the conventional culture method and identified based on the morphological characteristics, BOX-PCR fingerprints, and molecular sequences. Furthermore, the clustering analysis of different strains was conducted. Sporangial inhibition was tested with equal volumes of strain suspension or fermentation mixed with the sporangial suspension of P. viticola, and the control effects of isolates and their sterile fermentation against grape downy mildew were tested on detached grape leaves. [Results] A total of 90 bacterial strains and 110 fungal strains were isolated, belonging to eight bacterial genera and 14 fungal genera, respectively. The pathosphere of P. viticola in the same province and the same year exhibited similar microbial community composition. Notably, strains of Pseudomonas spp. and Cladosporium spp. exhibited stable populations on grape cultivars collected from different provinces. A majority (over 80.0%) of strains with stable populations in two consecutive years demonstrated significant biocontrol effects against grape downy mildew. Six Acremonium strains with ubiquitous distribution demonstrated the biocontrol effect up to 100.0%. Sterile fermentation of the fungal strains Aspergillus niger NX2F, Thecaphora amaranthi BJ1G, and Rhizopus stolonifer BM1L showed the control effects of 100.0% against grape downy mildew. [Conclusion] The culturable bacterial and fungal communities in the pathosphere of P. viticola were mainly affected by geographical factors in different provinces, and most of the culturable microorganisms presented stable and strong biocontrol effects on grape downy mildew. To the best of our knowledge, it is the first comprehensive report that Acremonium spp. were epibiotic fungi and consistently associated with P. viticola, providing rich and valuable biocontrol resources for grape downy mildew.

Abstract:[Objective] Pseudoalteromonas, an endemic and dominant genus in the ocean, possesses multiple methyl-accepting chemotaxis proteins (MCPs), the functions of which remain unclear. [Methods] The chemotaxis of Pseudoalteromonas arabiensis N1230-9 isolated from surface seawater of the Pacific Ocean towards 23 carbon sources was analyzed by a plate-based assay for swimming motility. Two sCache domain-containing genes (woc28264 and woc27036) encoding MCPs were deleted by homologous recombination. The chemotaxis of the two mutants to 10 carbon sources was then analyzed. [Results] Strain N1230-9 showed obvious chemotaxis to 10 carbon sources: trehalose, maltose, sucrose, N-acetylglucosamine, l-malic acid, sodium acetate, sodium propionate, sodium pyruvate, citric acid, and succinic acid. WOC28264 was a specific chemotactic receptor for l-malic acid and sucrose, while WOC27036 was a specific chemotactic receptor for citric acid and succinic acid. Both WOC28264 and WOC27036 were chemotactic receptors for N-acetylglucosamine and trehalose. [Conclusion] WOC28264 and WOC27036 have overlapping carbon source effectors.

Abstract:[Objective] To investigate the community structure, diversity, and plant-growth- promoting functions of endophytic bacteria in the protocorms of Epidendrum sp. and mine the core bacterial groups associated with the seed germination. [Methods] We collected five samples including three types of protocorms (germinated on MS1 medium, pine bark, and rotten oak leaves) and two types of substrates (pine bark and rotten oak leaves). High-throughput sequencing of the 16S rRNA gene was employed to compare the diversity and community composition of bacterial communities under different germination conditions. The conventional method was used to isolate the endophytic bacteria from the protocorms germinated on pine bark and rotten oak leaves (i.e., from symbiotic seed germination), and the growth-promoting potential of the isolates was evaluated. [Results] A total of 2 735 operational taxonomic units (OTUs) were obtained from the five samples, belonging to 876 genera, 453 families of 41 phyla, among which Proteobacteria and Actinobacteria were the dominant phyla. The results of principal coordinates analysis showed that there were differences in the bacterial community structure between the protocorms of Epidendrum sp. and the substrates, and the community structure of endophytic bacteria in the protocorms germinated on the MS1 medium was closest to that on pine bark. Functional prediction indicated that the endophytic bacteria in the protocorms germinated on rotten oak leaves had higher nitrogen-fixing ability than that in other types of protocorms. Nineteen isolates were obtained from protocorms geminated on pine bark and rotten oak leaves, belonging to 16 species of 12 genera. Tumebacillus flagellatus, Bradyrhizobium cenepequi, and Weizmannia ginsengihumi were the common species in the protocorms germinated on pine bark and rotten oak leaves. Pseudomonas koreensis and W. ginsengihumi had the potential of solubilizing phosphorus and producing indole-3-acetic acid (IAA) and siderophores. [Conclusion] The Epidendrum sp. protocorms germinated in different environments harbor rich endophytic bacteria. The endophytic bacteria isolated from the protocorms from symbiotic seed germination had plant growth-promoting functions, such as fixing nitrogen, solubilizing phosphorus, and producing IAA and siderophores. This study provides a scientific basis for mining the microbial resources related to seed germination of orchids and studying the interactions between orchids and microorganisms.

Abstract:[Background] Bacteria of Enterobacteriaceae are the main pathogens of foodborne and clinical infections, posing a threat to human and animal health. The numerical identification method using API 20E as the “gold standard” is one of the main identification methods for Enterobacteriaceae. However, the existing numerical identification methods have problems such as laborious sample addition, low accuracy, and high prices. [Objective] To develop a semi-automatic, high-precision, and low-cost biochemical kit for identifying the bacteria of Enterobacteriaceae based on the numerical identification method. [Methods] On the basis of the theoretical model and supporting software of the numerical identification system for Enterobacteriaceae established by our team, we designed and optimized 24 biochemical matrix trace formulations. Furthermore, a semi-automatic freeze-drying identification strip was developed. Using the commercialized numerical identification strip API 20E, MALDI-TOF MS, and 16S rRNA gene sequencing as the controls, we evaluated the performance of the developed biochemical kit that integrated semi-automatic biochemical identification strips and online analysis software. [Results] The biochemical spectra of 458 strains of Enterobacteriaceae were obtained, with the overall identification accuracy of 98.5% at the genus level and 96.5% at the species level. The kit only needed twice sample addition to obtain the identification results, with the price only 4.46% that of API 20E. The shelf life of the product was 7 days, and the biochemical experiments had repeatability. [Conclusion] We develop a semi-automatic biochemical kit for identification of Enterobacteriaceae based on the numerical identification method, which has simple operation, a low cost, and high accuracy. This study provides technical support for the identification of Enterobacteriaceae and the development of biochemical products for numerical identification of other families and genera of bacteria.