Abstract:[Background] As an important energy resource, coal is in high demand in production activities. However, long-term coal mining causes serious damage to the eco-environment. Microbial remediation, as an environmentally friendly, simple, and low-cost remediation method, has broad application prospects in the restoration of coal mining areas. [Objective] We used a composite microbial agent for the remediation of spoil sites in coal mining areas, aiming to restore the fragmented eco-environment of coal mines through microbial activities and interactions of microorganisms with plant roots. [Methods] Utilizing 16S rRNA gene high-throughput sequencing, we analyzed the microbial community structure at the remediation site. By monitoring the soil nutrient content, heavy metal concentrations, and growth of buckwheat plants, we assessed the efficacy of microbial remediation and explored the functions of the microorganisms. [Results] There were diverse microbial species with remediation capabilities at the experimental area. The addition of the microbial agent reduced the microbial diversity but had a minor impact on the indigenous microbial community structure at the experimental site. The on-site restoration for 150 days increased the organic matter, total nitrogen, total potassium, available nitrogen, available phosphorus, and available potassium by 70%, 20%, 48.4%, 40%, 26.8%, and 24.2%, respectively, which indicated the significant recovery in soil fertility. The organic matter, available phosphorus, and available potassium showed an increasing trend during the restoration period, indicating that functional microorganisms continuously provided efficient nutrients for plant growth. The decrease of 49% in arsenic and 41% in copper indicated that microorganisms played a role in the remediation of heavy metals at the site. Microorganisms promoted the growth of plant roots, enhancing the absorption and utilization of nutrients by plants. They increased the buckwheat plant height, dry weight, and crude protein content by 30%, 100%, and 22.4%, respectively. [Conclusion] The microbial agent significantly improved the soil fertility, suppressed the toxic effects of heavy metals in the soil, and promoted the growth of economic crops at the experimental site. Microbial remediation holds a great application value in the restoration of fragmented ecosystems in coal mining areas.

Abstract:[Background] In August and September 2022, the plant samples of leaf blotch and head rot were collected from sporadic diseased fields of confectionery sunflower (Helianthus annuus) in Lanzhou, Gansu, China. [Objective] To identify and characterize the pathogens of leaf blotch and head rot. [Methods] We isolated the pathogens by the single spore isolation method, measured the pathogenicity of the isolates based on Koch's postulates, and identified the isolates by morphological observation and molecular biological methods. Furthermore, we determined the optimum growth temperatures of the isolates by the plate culture method, and the changes of sclerotium formation characteristics of the tested isolates were observed after cultured at 30℃ (growth inhibition temperature). [Results] Twelve Botrytis isolates with different colony morphology were isolated from the diseased plant samples. The inoculation of four isolates, SKY-A to SKY-D, on detached sunflower leaves and heads induced the symptoms similar to those of natural diseases in the field, and the re-isolation rates of inoculated isolates from the infected leaves and heads were 100%. When the four isolates were cultured on PDA plates at 15℃ and 20℃ for 10–40 d, macroconidia were pale to light brown, unicellular, oval, oblong, spherical, subspherical, clavate or irregular shaped, (6.0–14.2 (–20.1)) μm×(6.0–10.4 (–14.9)) μm. The sclerotia were superficial, black, spherical, subspherical, ellipse or irregularly shaped, (0.5–11.1) mm×(0.5–5.0) mm. Type-I microsclerotia were reddish brown to dark brown, superficial or submerged, (26.9–492.5) μm×(14.9–149.3) μm, with the cell sizes of (12.7–35.0) μm×(11.9–25.3) μm. Type-II microsclerotia were nearly black, submerged, (35.8–373.1) μm×(23.9–229.9) μm, with the cell sizes of (8.2–16.4) μm×(8.2–14.9) μm. Pseudo-microsclerotia consisting of specialized appressoria were dark brown to nearly black, submerged, (32.0–447.8) μm×(19.4–358.2) μm. The optimal temperature range for mycelial growth and the temperature range for sclerotium formation were 20–25℃ and 5–25℃, respectively. After incubation at 30℃ for 7 d and then at 20℃ for 14 d, SKY-B and SKY-C originally uncapable of forming sclerotia formed sclerotia. Moreover, the mutants retained the ability of forming sclerotia in subculturing. BLASTn analysis showed that the rDNA-ITS sequences of SKY-A to SKY-D (National Microbiology Data Center Acc. No. NMDCN00038NP–NMDCN00038NS) had the similarity of 99.8%–100.0% with those of Botrytis cinerea strains in GenBank. The phylogenetic analysis based on the glyceraldehyde 3-phosphate dehydrogenase gene (g3pdh), heat shock protein 60 gene (hsp60), and DNA-dependent RNA polymerase subunit II gene (rpb2) showed that the four isolates were clustered in different subgroups of B. cinerea. [Conclusion] The pathogens causing leaf blotch and head rot on confectionery sunflower were identified as B. cinerea. This is the first report of leaf blotch and head rot caused by B. cinerea on confectionery sunflower in the Northwest China. For the first time, we discover that B. cinerea can produce two types of microsclerotia. The heat stress at 30℃ induced the B. cinerea isolates uncapable of forming sclerotia to form sclerotia, and the sclerotium-forming ability of the mutants could be inherited.

Abstract:The application of gene editing enables the efficient and accurate genome modification of filamentous fungi. However, the actual operation is influenced by a variety of factors, which result in low efficiency of precise gene editing mediated by homologous recombination. In this review, we summarize the current methods to improve the precision editing efficiency of filamentous fungi, so as to improve the application of gene editing in strain modification.

Abstract:Quorum sensing is a communication mechanism between microbial populations. By recognizing signal molecules, microorganisms can exchange information within and outside the population and regulate their physiological behaviors. Quorum quenching can block quorum sensing by interrupting the synthesis, secretion, and recognition of quorum-sensing signal molecules. In the interaction process between microalgae and bacteria, quorum sensing and quorum quenching jointly regulates the physiological changes in microalgae and bacteria for their balanced coexistence. This paper introduces the occurrence process and detection of quorum sensing and elaborates on the role of quorum sensing in the interaction between microalgae and bacteria. Furthermore, this paper expounds the occurrence mechanism of quorum quenching and the performance and ecological benefits of quorum quenching in the interaction between microalgae and bacteria. Finally, prospects are put forward for studying the mechanism of the interaction between microalgae and bacteria and the role of the interaction in marine ecosystems.

Abstract:The type IV secretion systems (T4SSs) are ubiquitous and versatile in bacteria, capable of transporting proteins, DNA, and protein-DNA complexes. Bacterial conjugation is a key mechanism of horizontal gene transfer. T4SSs can mediate the transfer of conjugative plasmids or integrative elements from one bacterium to another, which endow bacteria with new genes and functions. The acquisition of genes in this way not only enables bacteria to gain new functions but also helps them occupy niches, promoting the adaptation of bacteria to a new or changing environment. This article summarizes the roles of T4SSs, especially in bacterial adaptation to heavy metal-polluted environments.

Abstract:Postmortem interval (PMI) estimation is one of the important contents in forensic identification. In recent years, with the development of high-throughput sequencing, bioinformatics, and machine learning, the changes of microbial community structure provide a new possibility for PMI estimation. The evolution pattern of the microbial community in the process of decomposition can be used to predict PMI. From the nature of cadaver decomposition, we summarize the examination methods and applications of cadaver microbial changes, and discuss the problems and prospects of using the microbial community changes to predict PMI.

Abstract:Brucellosis, caused by the bacteria of Brucella, is considered one of the major zoonotic diseases worldwide. As the key virulence factors of Brucella, outer membrane proteins not only play a vital role in the host immune process against Brucella but also are potential immunogens and protective antigens of Brucella. These proteins can stimulate strong immune response of the host and be used as diagnostic antigens and targets of novel vaccines against Brucella. We reviewed the research progress in the immunogenicity of key outer membrane proteins of Brucella, aiming to provide a reference for laboratory diagnosis and new vaccine development against Brucella.

Abstract:Recombinase polymerase amplification (RPA) is a technology that utilizes proteins such as recombinase (T4 UvsX), single-stranded DNA binding protein (T4 Gp32), and strand replacement DNA polymerase (Bsu) to amplify target nucleic acids at constant temperatures. In recent years, RPA plays a unique role in the detection of bacteria, viruses, parasites, mycoplasma, and chlamydia for it can amplify the target nucleic acid fragment with constant temperature and high efficiency, it gradually becomes a key technology for point-of-care testing (POCT). This review takes the principle of RPA technology as a starting point, it describes the clinical application, shortcomings, and improvement of RPA, and RPA is expected to provide a new idea for the rapid detection and diagnosis of clinical diseases.

Abstract:[Background] (S)-Acetoin is a class of important fine chemicals with wide applications. Efficient and sustainable synthesis of (S)-acetoin is of special interest. [Objective] Due to the high thermostability, ancestral carbonyl reductases are promising in industrial biocatalysis. This study aims to mine an ancestral carbonyl reductase with high activity and thermostability, characterize its enzymatic properties, and establish an efficient enzymatic catalysis system for the synthesis of (S)-acetoin. [Methods] The gene mining strategy based on ancestral sequence reconstruction was adopted to identify ancestral carbonyl reductases. The enzyme properties were characterized, and the purified ancestral carbonyl reductase was used for the establishment of the (S)-acetoin synthesis system. [Results] An ancestral carbonyl reductase AncBDH1 was identified and heterogeneously expressed in Escherichia coli in the soluble form, and it displayed a high activity in the asymmetric reduction of diethyl into (S)-acetoin. The sequence of AncBDH1 contained the conserved motifs of short-chain dehydrogenase/reductase superfamily and exhibited low similarity with the sequences of other reported carbonyl reductases. AncBDH1 was purified by nickel-affinity chromatography for enzyme characterization. It displayed high dependence on NADH instead of NADPH as a cofactor, with the optimum performance at pH 7.0 and 60℃. The activity of AncBDH1 was not dependent on metal ions and AncBDH1 could tolerate organic solvents such as dimethyl sulfoxide. AncBDH1 completely reduced 200 mmol/L diethyl into (S)-acetoin within 12 h. [Conclusion] This study provides a novel ancestral carbonyl reductase AncBDH1 with tolerance to high temperature and organic solvents and capable of catalyzing the synthesis of (S)-acetoin, laying a certain research foundation for the biocatalytic synthesis of (S)-acetoin.

Abstract:[Background] Glutamate decarboxylase is widely present in organisms. γ-aminobutyric acid, the catalytic product of glutamate decarboxylase, is an inhibitory neurotransmitter in mammals. At present, many glutamate decarboxylases from microorganisms have poor thermal stability and acid-base stability. [Objective] To heterologously express a glutamate decarboxylase gene mined from gut microbiota and study the enzymatic properties of the recombinant protein, so as to provide an enzyme source for the biosynthesis of γ-aminobutyric acid. [Methods] The glutamate decarboxylase gene was amplified from the fecal microbial metagenome of Pygmy loris and expressed in Escherichia coli BL21(DE3). The enzymatic properties of the recombinant enzyme were characterized, and the enzyme was then used for the whole-cell synthesis of γ-aminobutyric acid. [Results] The glutamate decarboxylase gene Xby1_8 was obtained, and the molecular weight of the recombinant enzyme Xby1_8 was 54.46 kDa. Under optimal reaction conditions of pH 5.0 and 55℃, the K_m andV_max values of the enzyme were (10.2±1.5) mmol/L and (3 574.0±198.3) μmol/(min·mg), respectively. Xby1_8 had the highest specific activity of 3 106.2 U/mg compared with other glutamate decarboxylases from microbial sources. Xby1_8 had good pH and thermal stability. The residual activity of Xby1_8 was still more than 100% after incubation at pH 4.0–8.0 or 30–50℃ for 1 h. The whole-cell recombinant E. coli/Xby1_8 (OD₆₀₀ of 3.5) reacted with 260 mmol/L l-glutamic acid at 55℃ for 2.5 h showed the conversion rate of 100% for the production of γ-aminobutyric acid. [Conclusion] The glutamate decarboxylase gene Xby1_8 was obtained from the fecal microbial metagenome and successfully expressed in E. coli BL21(DE3). This enzyme has good pH and thermal stability. The whole cell preparation of γ-aminobutyric acid from recombinant E. coli/Xby1_8 has a high conversion rate, thereby demonstrating promising potential for application in food and industrial fields.

Abstract:[Background] Glutaminase is an enzyme which can catalyzes the hydrolysis of l-glutamine to l-glutamic acid and ammonia, and it has been well used in the food and medicine fields. [Objective] To study the influences of different promoters and signal peptides on the secretory expression of glutaminase and construct recombinant Bacillus subtilis strains with efficient secretory expression of glutaminase to improve the production of glutaminase. [Methods] B. subtilis SCK6 was selected as the host, and the glutaminase expression vectors and recombinant strains with different promoters and signal peptides were constructed. The effects of different gene elements on the expression and secretion of glutaminase were studied, and the optimal promoter and signal peptide were selected and combined to construct an efficient and robust glutaminase-producing strain. [Results] A recombinant strain with efficient secretory expression of glutaminase was constructed. With P_HpaII-P_aprE as the promoter and YndA as the signal peptide, the strain showed the extracellular glutaminase activity reaching 6.7 U/mL in shake flask fermentation. [Conclusion] By screening and combining the promoter and signal peptide, we constructed a recombinant strain with robust production of glutaminase, providing an effective method for the efficient secretory expression of enzymes.

Abstract:[Background] The alkali metal sodium affects coal burning equipment and gasification efficiency in the coal combustion process and acid wastewater is produced in the pickling process. [Objective] To propose a novel method for removing organic sodium from coal by microorganisms. [Methods] A bacterial strain, which was isolated and screened out from Huangqihai, Chahar Right Front Banner, Ulanqab, Inner Mongolia, was identified based on morphological, physiological, and biochemical characteristics and the 16S rRNA gene sequence, and a phylogenetic tree was constructed. The optimal growth conditions of the strain were investigated by single factor tests, and the growth curve was plotted. The optimal removal conditions of organic sodium in coal by the strain were determined, and the removal effect was evaluated under the optimal conditions. [Results] A strain that can remove organic sodium from coal was screened out and identified as Sphingomonas aquatilis. The strain showed the best growth performance in the medium with glucose (4 g/L) as the carbon source, peptone (13 g/L) as the nitrogen source, and initial pH 8.0 when it was cultured at 30℃ and 150 r/min with the inoculum quantity of 3% and liquid loading of 100 mL. The optimal removal conditions of organic sodium from coal were as follows:initial pH 8.0, liquid loading of 80 mL, coal slurry concentration of 5 g/L, and inoculum quantity of 7%. Under the conditions, the removal efficiency of organic sodium from coal by strain CSW-1 was 23.79%. [Conclusion] A novel method for removing organic sodium from coal by microorganisms is proposed for the first time, which provides a new idea for the efficient and clean utilization of coal.

Abstract:[Background] Mining practices has a serious impact on the eco-environment around the mine. It is of great significance to explore the characteristics of soil nutrients and bacterial community structure around the mine for vegetation restoration. [Objective] To explore the characteristics of soil nutrients and bacterial community structures in different habitats around the Pulang Copper Mine in Shangri-La, Yunnan province. [Methods] The rhizosphere soil samples of dominant herbaceous plants were collected from four habitats (subalpine meadow, valley meadow, mine woodland, and subalpine woodland) around the Pulang Copper Mine to reveal the characteristics of soil nutrients and bacterial community structures of different habitats around the mine. [Results] The soil samples from the mine woodland had the highest nutrient content, which generally reached the medium or above level. Acidobacteriota, Actinobacteriota, and Proteobacteria were the dominant phyla of soil bacteria in the four habitats around the mine, with relative abundance ranging from 11.7% to 60.4%. Bradyrhizobium, Afipia, and Arthrobacter were the dominant genera, with relative abundance ranging from 6.1% to 53.6%. Mantel test results showed that soil organic matter had significant effects on the soil bacterial community structure at the genus level. [Conclusion] The results suggested that soil bacterial abundance was higher in summer than in winter (P<0.05), while there were no significant differences in soil bacterial relative abundance and diversity between the four habitats around the Pulang Copper Mine in summer and winter (P>0.05). The findings contribute to the understanding of mine ecosystems and provide a theoretical basis for the ecological restoration and soil pollution treatment in mining areas.

Abstract:[Background] Some metal-tolerant microorganisms have been found to reduce the availability of metals in soil, reduce the transportation and accumulation of metals in plants, and promote plant growth. With low cost-effectiveness and environmental friendliness, these microorganisms demonstrate promising prospects for applications in agriculture, industry, and the environment. [Objective] To isolate and identify high-quality strains tolerant to Cd(II), examine the morphological characteristics and Cd(II) tolerance of the strains, analyze the molecular genetic characteristics of the strains at the genomic level, mine their potential functions and Cd(II) tolerance-related genes, and observe the effects of the strains on wheat seed germination under cadmium stress. [Methods] Over ten soil samples were collected from phosphate and coal mining areas in Deyang City, Sichuan Province. A Cd(II)-tolerant strain was isolated in selective culture media, and its morphological characteristics were observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The Cd(II) tolerance of the strain was examined based on the OD₆₀₀. Whole-genome sequencing was performed using Illumina NovaSeq and Oxford Nanopore ONT platforms for identification and genetic function analysis of the strain. Bioinformatics tools were used for genetic assembly, functional annotation, and mining of genes related to Cd(II) adaptation. The wheat seed germination rate, the bud and root growth of wheat seedlings under different treatments were determined to reveal the germination characteristics of wheat seeds under Cd(II) stress. [Results] One Cd(II)-tolerant strain was isolated, identified as Exiguobacterium acetylicum and named TC13. The whole-genome sequence assembly of TC13 resulted in one chromosome and one circular plasmid with the lengths of 3 192 165 bp and 167 401 bp and the G+C content of 46.62% and 44.63%, respectively. The chromosome and circular plasmid carried 3 231 and 154 DNA coding sequences, and the chromosome contained 54 ncRNAs. Functional gene database alignment results revealed that multiple genes were involved in biological processes such as heavy metal transport, efflux, and stress response in the strain, which may be associated with its cadmium tolerance. In addition, the strain increased the germination rate of wheat seeds and improved the growth of wheat seedlings under Cd(II) stress.[Conclusion] This study explored the genome structure of E. acetylicum TC13 by whole-genome sequencing, enriching the genetic information of strains of the same species, revealing the Cd(II) tolerance-related genes and confirmed the strain TC13 could promot the growth of wheat grain under Cd(II) stress. The strain TC13 has potential application prospects in soil remediation and plant growth promotion.

Abstract:[Background] In recent years, cosmetic recall cases caused by contamination with objectionable microorganisms have kept emerging. The microbial detection indicators in the Cosmetic Safety Technical Specifications (2015 Edition) are not perfect and difficult to meet the needs of quality control and evaluation of cosmetics products. [Objective] To isolate and identify the contaminant microorganisms in a batch of facial masks with microorganisms out of limits in the national sampling inspection of cosmetics in 2022, explore the performance of different identification methods, determine whether the contaminant microorganisms are objectionable microorganisms or not, and evaluate the safety of the contaminant microorganisms.[Methods] According to the Cosmetic Safety Technical Specifications (2015 Edition), the contaminant microorganisms in the facial masks were isolated and identified by VITEK 2 system, matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and 16S rRNA gene sequencing. Furthermore, the safety evaluation and detection of virulence genes and drug resistance genes were carried out for the contaminant microorganisms. [Results] The results obtained with the three identification techniques all showed that the contaminant microorganism in the facial masks was an objectionable microorganism, Pluralibacter gergoviae. High-throughput sequencing detected 52 virulence genes and predicted 429 other virulence-related genes, which suggested that this bacterium had great safety risks. At the same time, 24 drug resistance genes were detected, suggesting that the bacterium may be resistant to 24 antibiotics such as penicillin, cephalosporin, vancomycin, tetracycline, and aminoglycosides. Moreover, 10 multi-drug resistance genes were detected, and the resistance mechanism was associated with the production of efflux pumps. It is suggested that the bacterium have a certain risk of multi-drug resistance. [Conclusion] In this study, an objectionable microorganism P. gergoviae was isolated from a batch of facial masks with microorganisms out of limit. This bacterium is capable of producing toxins and has a potential safety risk of multidrug resistance. It should be considered to include this bacterium into the microbial detection after risk assessment.

Abstract:[Background] Despite the abundant production, rice straw is rarely used as animal feed because of the difficulty in degrading lignocellulose. [Objective] To investigate the fungal community dynamics in rice straw over time and the degradation of lignocellulose by fungi in the early stage of microbial fermentation.[Methods] Rice straw was inoculated with 1.2×10⁶ CFU/g-DM of Bacillus licheniformis and 5×10⁶ CFU/g-DM of Lactobacillus plantarum for microbial fermentation. The ITS sequencing was performed to analyze the fungal community on days 1, 3, 7, and 15. [Results] The highest fungal diversity was observed on day 7. The dominant phyla during fermentation were Ascomycetes (64.2%) and Basidiomycota (30.2%). Compared with that on day 1, the relative abundance of Ascomycota increased on day 7and day 15 (P<0.001), and that of Basidiomycota decreased on day 7 and day 15 (P<0.001). The dominant genera included Hannaella (11.6%), Issatchenkia (8.1%), Kazachstania (6.3%), Saccharomyces (6.2%), Filobasidium (5.1%), Acremonium (2.4%), and Aspergillus (2.2%). Compared with that on day 1, the relative abundance of Hannaella decreased on day 15 (P<0.001) and that of Acremonium and Aspergillus increased on day 7 (P<0.001). PICRUSt predictions indicated that β-glucosidase, laccase, α-galactosidase, and β-mannosidase were the most abundant lignocellulose-degrading enzymes. The results of Spearman correlation analysis showed that Ascomycota had a positive correlation with β-mannosidase (P<0.05). Hannaella was positively correlated with β-glucosidase and β-mannosidase (P<0.05). In addition, both Acremonium and Aspergillus showed strong positive correlations with β-glucosidase, β-mannosidase, laccase, and α-galactosidase (P<0.001). [Conclusion] During the microbial fermentation of rice straw, fungi degrade the lignocellulose. This paper presents theoretical underpinnings for the efficient use of fungi in the microbial fermentation and the biodegradation of lignocellulose.

Abstract:[Background] The antioxidant system plays an important role in plant tolerance to stress, and whether rhizobia affect the antioxidant system of legumes to improve plant tolerance to stress remains unclear. [Objective] Pongamia pinnata with strong stress tolerance was selected as the representative of legumes in this study, and the pot experiments of inoculating Rhizobium sp. PP3 to P. pinnata under nickel stress were performed. [Methods] The activities of four antioxidant enzymes and the content of four non-enzymatic antioxidants in the roots, stems and leaves of P. pinnata in different treatments were determined by the enzyme linked immunosorbent assay. [Results] The activities of antioxidant enzymes in P. pinnata followed the trend of superoxide dismutase (SOD)>peroxidase (POD)>catalase (CAT)>glutathione reductase (GR), and the content non-enzymatic antioxidants was in the order of glutathione (GSH)>flavonoid (FLA)>oligomeric proantho cyanidins (OPC)>ascorbic acid (AsA). In the case of no nitrogen supply and 40 mg/L Ni²⁺ stress, Rhizobium sp. PP3 increased the POD activity in the stems by 61.8%, and the SOD, GR, and CAT activities in the leaves by 5.7%, 23.6%, and 36.1%, respectively. Under the stress of nitrogen and 40 mg/L Ni²⁺, Rhizobium sp. PP3 increased the content of GSH, FLA, and OPC in the roots by 55.9%, 61.6%, and 38.6%, respectively, and the GSH content in the leaves by 14.8% under the stress of 40 mg/L Ni²⁺. [Conclusion] Rhizobia had significant effects on the four antioxidant enzymes and non-enzymatic antioxidants in P. pinnata, while the effects on antioxidant enzymes and antioxidants varied in different tissues. The results indicate that rhizobia regulate the antioxidant system of P. pinnata in response to nickel stress, and they play a role in strengthening the stress tolerance of P. pinnata in the nickel-contaminated soil.

Abstract:[Background] In recent years, mining antimicrobial components from the fermentation products of endophytes has become an important way to discover new bioactive components. [Objective] To find new endophytes with high antimicrobial activity, explore the optimum fermentation conditions for the production of bioactive components, and decipher their antimicrobial mechanisms. [Methods] The endophytes were isolated from lemon peels and seeds by tissue culture and streaking method. The strains with strong inhibitory effects on pathogenic microorganisms were screened by filter paper and Oxford cup methods and then identified by morphological observation and 16S rRNA gene sequencing. The antimicrobial effect of the fermentation liquid extract (B02c) of the active strain was examined by the microplate method. Single factor tests and response surface experiment were carried out to optimize the fermentation conditions. Propidium iodide/SYTO-9 probe marking combined with flow cytometry were employed to measure the effect of B02c on the cell membrane of the tested strains. [Results] A strain Z10 with significant inhibitory effect on Staphylococcus aureus was isolated from lemon seeds and identified as Bacillus sp. The optimal fermentation conditions of strain Z10 were as follows:addition of orange peel residue at 1.6 g/100 mL, pH 6.8, inoculum amount of 10.0%, rotation speed of 172 r/min, and culture for 36 h. After optimization, the obtained B02c showed the inhibition rate reaching 84.36%, increased by 16.36% compared with that of the fermentation liquid obtained at the original conditions. B02c showed the MIC of 3.125 mg/mL against S. aureus and destroyed the cell membrane integrity of S. aureus at this dose. [Conclusion] B02c of the lemon endophyte Z10 under optimized fermentation conditions demonstrates a strong inhibitory effect on S. aureus, serving the screening and industrial production of new and efficient natural bioactive substances.

Abstract:[Background] Saussurea involucrata is a rare and endangered medicinal plant growing in the alpine region. However, little is known about the cold adaptation of endophytic fungi (EFs) isolated from S. involucrata. [Objective] To well understand the diversity and biological characteristics of EFs isolated from S. involucrata roots. [Methods] EFs were isolated by tissue block culture at 4℃ and 15℃, respectively. All the strains were preliminarily identified based on the ITS sequences. According to the ITS sequence similarity, strains were clustered into OTU for the diversity analysis. The representative strain (RS) of each OTU was selected, and the morphology of RSs was observed. The growth rates of RSs and the effects of RSs on S. involucrata seedling growth were investigated at different temperatures. [Results] A total of 142 strains of EFs were isolated, including 68 and 74 strains obtained at 4℃ and 15℃, respectively. The 142 strains were clustered into 14 OTUs, which belonged to 9 genera, 7 families, 5 orders, and 4 classes of 2 phyla. At 4℃ and 15℃, respectively, 10 and 11 OTUs were obtained and indicated different taxa (P<0.01). The community at 4℃ included 3 unique OTUs, in which Alternaria was the dominant genus. The community at 15℃ included 4 unique OTUs, in which Rhexocercosporidium was the dominant genus. Ten strains of dark septate endophytes (DSEs) were identified from 14 RSs, and 9 of them were psychrophiles or psychrotrophs. There were 6 RSs that could promote the growth of S. involucrata seedlings, 5 of which were DSEs, including 3 psychrotrophs and 2 psychrophiles. S11 and S12 promoted the growth of seedlings at both 15℃ and 20℃, while S01, S13, and S14 promoted the seedling growth only at 15℃. S13 and S14, the RSs of dominant Rhexocercosporidium, were DSEs and psychrotrophs capable of promoting plant growth. The plant growth-promoting effects of S01 and S12 at 15℃ were stronger than those at 20℃. Compared with those at 20℃, S01 and S12 increased the total root length of seedlings cultured at 15℃ by 2.17‒2.27 times (P<0.05). [Conclusion] The EFs isolated at 4℃ and 15℃ showed rich diversity. The isolation temperature affected the composition and structure of fungal communities. Lower temperatures were beneficial to the isolation of cold-adaptable DSEs.

Abstract:[Background] Fungi play an important role in the flavor and texture of Paocai and influence the shelf life of Paocai and its brine. Brine is the traditional method of Paocai production in China, and it is also the key for maintaining the original flavor of Paocai. [Objective] To analyze the dynamics of the fungal community and its relationship with physicochemical factors of Paocai brine in sealed storage. [Methods] The amplicon sequence variants (ASVs) were employed to monitor the dynamics of the fungal community in Paocai brine stored at 25℃ for 30 d. A flow analyzer was used to reveal the changes in physicochemical factors of the brine. [Results] According to the fungal community dynamics and physicochemical factor changes in the Paocai brine, the fungal community succession within 30 d can be divided into three stages (early:1–5 d; middle:6–16 d; late:18–30 d). A total of 461 fungal genera belonging to 226 families, 98 orders, 39 classes of 10 phyla were annotated in the three stages. In the early stage, the fungal composition was complex and changing, and the interactions were strongly influenced by pH and total nitrogen (TN). The middle stage presented changed dominant genera and increased rare genera, and the interactions were weakened and regulated by TN. In the late stage, the number of rare genera kept decreasing, and the community structure was simple, with weak interactions which were influenced by nitrate nitrogen (NN), phosphate (P), and total phosphorus (TP). [Conclusion] The fungal community in the Paocai brine stored at 25℃ for 30 d varied in the early, middle, and late stages. The Paocai brine stored at 25℃ for 30 d was safe to be used within one week and could guarantee the flavor of Paocai. Moreover, cold chain storage could adequately extend the shelf life. It is worth exploring the rich fungal resources in the Paocai system.

Abstract:[Background] Integrons, as key mobile genetic elements associated with the transmission of antibiotic resistance genes (ARGs), can capture and express ARGs through site-specific recombination, playing a role in the transmission of bacterial antimicrobial resistance (AMR). Therefore, it is of great significance to analyze the integron-carrying status of Escherichia coli from aquatic food and clarify the status of integron-mediated multi-drug resistance (MDR) of E. coli for AMR monitoring and antibiotic use guidance in aquaculture. [Objective] To analyze the diversity of class I, II, and III integrons of E. coli in aquatic food. [Methods] One hundred and sixty samples of Pacific white shrimp (Penaeus vannamei), 160 samples of Pacific oyster (Crassostrea gigas), and 160 samples of Pacific mackerel (Pneumatophorus japonicas) were collected from a farmer's market in Zhejiang province. E. coli was isolated and identified by Eosin Methylene Bule Agar and PCR method. The AMR characteristics of E. coli to 9 categories of 19 antibiotics were analyzed by the Kirby-Bauer disc diffusion method. The integrons and diversity of gene cassettes (GCs) carried by E. coli from aquatic products were analyzed by PCR. [Results] Fifteen, 59, and 26 strains of E. coli were isolated from the samples of Pacific white shrimp, Pacific oyster, and Pacific mackerel, respectively, with a total of 100 strains. The MDR rates of E. coli isolates from Pacific white shrimp, Pacific oyster, and Pacific mackerel were 93.3% (14/15), 76.3% (45/59), and 80.8% (21/26), respectively. The class I, II, and III integron-carrying rates of E. coli isolates was 71.0% (71/100), 5.0% (5/100), and 0.0%, respectively. A total of 10 different class I integron GC arrays and three different class II integron GC arrays were detected. The class II integron GC array dfrA1-catB2-sat2-aadA1 was identified in E. coli for the first time. The Pearson correlation analysis showed that there was a positive correlation between the integron-carrying rate and MDR of E. coli from aquatic food (r=0.99, p<0.05). [Conclusion] This study has significance for revealing the integron-mediated transmission mechanism of ARGs of aquatic food-derived pathogenic microorganisms and promoting the healthy development of aquaculture.

Abstract:[Background] Bovine tuberculosis is a zoonotic infectious disease mainly caused by Mycobacterium bovis, which hinders the development of breeding industry and causes huge economic burden worldwide. [Objective] To prepare monoclonal antibodies against Mycobacterium tuberculosis CFP-10 protein and establish a competitive ELISA for the detection of bovine tuberculosis. [Methods] With rHis-CFP-10 protein as the immunogen, the mouse hybridoma fusion technique was employed to establish stable hybridoma cells that secreted antibodies. A monoclonal antibody was obtained from the hybridoma cell line 8E6 and subsequently conjugated with horseradish peroxidase after purification. A competitive ELISA for the detection of bovine tuberculosis was established based on the anti-CFP-10 monoclonal antibody. To assess the applicability of the established method, we employed the commercial BOVIGAM^TM kit and the established competitive ELISA assay to detect 155 dairy cows. [Results] A hybridoma cell line 8E6 stably secreting specific antibodies against CFP-10 was obtained. A competitive ELISA method was established based on the HRP-8E6 antibody. The working concentration of the coating antigen CFP-10-ESAT-6 and the dilution factor of HRP-8E6 were 0.75 μg/mL and 1:8 000, respectively. The established method showed the Cut-off of 47.10%, the limit of detection of 0.800 μg/mL, and good reproducibility (inter and intra-batch coefficients of variance being below 10%). The established method showed the sensitivity of 54.55% and the specificity of 100.00% in the detection of the serum samples from cattle with bovine tuberculosis and healthy cattle. In the clinical trial, the established method showed a positive coincidence rate of 78.57%, a negative coincidence rate of 82.35%, and a total coincidence rate of 80.65% compared with the BOVIGAM^TM kit. [Conclusion] An anti-CFP-10 antibody was successfully prepared and a competitive ELISA method was established using a CFP-10-ESAT-6 fusion protein with high specificity. The method is accurate and reliable and can be preliminarily applied to the detection of bovine tuberculosis.

Abstract:[Background] As an important group of rare actinomycetes, Amycolatopsis serves as a significant source of antimicrobial and anticancer drugs. The rich biosynthetic gene clusters (BGCs) of secondary metabolites in the genomes of Amycolatopsis await systematic analysis and mining. [Objective] To unveil the biosynthetic potential of secondary metabolites with different structural types in Amycolatopsis and establish genetic manipulation systems for two representative strains, laying a foundation for mining structurally novel or bioactive secondary metabolites. [Methods] The multi-locus sequence analysis (MLSA) was performed to evaluate the similarity among publicly available Amycolatopsis genomes. Simultaneously, antiSMASH was employed to analyze the gene clusters in all genomes and predict the structural information of the products. BiG-SCAPE was used for cluster analysis. The integration plasmid pSET152 was employed to optimize the conjugation transfer conditions for two representative Amycolatopsis strains, and then genetic manipulation systems were established. CRISPR-cBEST and homologous recombination were employed to determine the strategies for target gene inactivation. [Results] Bioinformatics analysis of 146 Amycolatopsis genomes revealed an average of 33 BGCs per genome. The BGCs associated with peptides, polyketides, and terpenes exhibited high abundance, with the majority differing from known BGCs. The optimal conjugation transfer conditions for two representative Amycolatopsis strains were successfully determined. Although CRISPR gene editing plasmids could be transferred into Amycolatopsis, none of the six vectors successfully edited the target gene. Conversely, target genes were successfully knocked out by homologous recombination, on the basis of which the gene editing systems were established. [Conclusion] The secondary metabolic potential unveils a rich repository of novel natural products within Amycolatopsis. Moreover, the successfully established genetic editing system enables the integration of exogenous genes and the inactivation of endogenous genes, laying a solid foundation for the mining and biosynthesis of novel secondary metabolites in this genus.

Abstract:[Background] Xinjiang is the main producing area of cotton, and Fusarium wilt seriously affects the cotton yield. However, the detailed pathogenesis of cotton Fusarium wilt remains to be reported. [Objective] To select the mutants with reduced pathogenicity from the Fusarium oxysporum mutant library and identity their genes conferring pathogenicity, so as to elucidate the molecular mechanism of the pathogen in causing Fusarium wilt in cotton. [Methods] On the basis of the previously established mutant library of F. oxysporum, By the identification of 136 randomly selected pathogenic mutants. Furthermore, we analyzed the phenotypes and flanking sequences. [Results] we screened out five mutants with significantly reduced pathogenicity, the morphology of mutant B-18 was different from that of the wild type, with purple colonies. The growth rate of the five mutants, especially B-18, decreased. In addition, the five mutants showed reduced spore production. After sequencing of the five mutants, Blast analysis was performed to determine the distribution of T-DNA in the pathogen genome. [Conclusion] From the five mutants with reduced pathogenicity, we identified the genes associated with the pathogenicity. The findings provide a solid theoretical basis for the functional research and cloning of the genes associated with the diseases in cotton.

Abstract:[Background] The mating types and mon-mon hybrids of edible mushrooms are usually identified based on the presence of clamp connections or not as observed by microscopy, which is time-consuming, labor-costing, and prone to false results. [Objective] A molecular-assisted breeding technique was established for identifying the monokaryon mating type and mon-mon hybrids of Lentinula edodes, aiming to provide technical support for improving the breeding efficiency. [Methods] Typing primers were designed based on the SNP loci in the conserved sequence of mating-type factors, and then allele specific (AS)-PCR was employed to identify the mating type of the single spore isolates of L. edodes strains L808 and YX7 and their mon-mon hybrids. [Results] Among the 38 single spore isolates of L808, there were 6, 13, 8, and 11 monokaryons of mating types A1B1, A2B2, A1B2, and A2B1, respectively. Among the 45 single spore isolates of YX7, there were 15, 8, 8, and 12 monokaryons of mating types A3B3, A4B4, A3B4, and A4B3, respectively, and 2 heterokaryons of the mating type A3A4B3B4. The 12 mon-mon hybrids included 10 heterozygotes and 2 non-heterozygotes. The identification results obtained based on the conventional method were consistent with those obtained by AS-PCR, except that the former was prone to misjudging heterokaryons as monokaryons. [Conclusion] The AS-PCR based on SNP loci can identify the monokaryon mating type and mon-mon hybrids of L. edodes and distinguish between monokaryons and heterokaryons, with high precision and efficiency. It serves as an ideal tool for molecular-assisted breeding of L. edodes.

Abstract:[Background] Oncolytic virus therapy is a new direction in the research on tumor treatment, and Newcastle disease virus (NDV) has attracted much attention because of its high biosafety and accurate targeting. However, viral clearance by neutralizing antibodies will weaken the sustained effect of oncolytic virus. The tumor cell-derived microvesicle delivery system is expected to break the above bottleneck. [Objective] To establish the tumor cell-derived microvesicle delivery system of oncolytic NDV and evaluate its antitumor effect in vitro. [Methods] Microvesicle-encapsulated NDV (MV@NDV) was extracted by differential centrifugation. The particle sizes of MV, NDV, and MV@NDV were measured by a particle size meter. The relative positions of MV and NDV in MV@NDV were observed by transmission electron microscopy. Western blotting (WB) was employed to determine the protein levels of Integrin β-1, Gpc 3, and Flotillin-1 of MV@NDV. Laser confocal microscopy was employed to observe MV@NDV 24 h post infection in Hepa 1-6 cells and the expression of NDV-HN protein in cells. An inverted microscope and the cell counting kit-8 (CCK-8) were used to examine the changes the morphology and viability of infected Hepa 1-6 cells 24, 48, and 72 h post infection, respectively. Quantitative real-time polymerase chain reaction (qPCR) and WB were employed to determine the mRNA levels of apoptosis-associated genes Caspase-3 and Caspase-9 and the protein level of Caspase-3, respectively, in the MV@NDV-infected Hepa 1-6 cells 24 h post infection. [Results] The particle sizes of MV, NDV, and MV@NDV were within the ranges of 141−342 nm, 91−825 nm, and 164−712 nm, respectively. WB results indicated that Integrin β-1, Gpc 3, and Flotillin-1 were expressed in MV@NDV. The Hepa 1-6 cells infected with MV@NDV and NDV showed red fluorescence and expressed NDV-HN protein. In addition, the viability of Hepa 1-6 cells decreased in the MV@NDV group compared with that in the NC group (P<0.05). The Hepa 1-6 cells in the MV@NDV group showed up-regulated mRNA levels of Caspase-3 and Caspase-9 and up-regulated protein level of Caspase-3 compared with the NC group (P<0.05). [Conclusion] The successful construction of NDV microvesicle delivery system MV@NDV is expected to solve the bottleneck of therapeutic oncolytic Newcastle disease virus (NDV) in the clinical translation, and provide new ideas and rationale for clinical NDV anti-tumor aspects.

Abstract:Farming-and-reading education plays a vital role in carrying forward Chinese traditional culture and driving the educational reform in agriculture-related colleges and universities to achieve the integration of moral, intellectual, physical, aesthetic, and labor education (quality education in five aspects). The supervisors' practice was taken as representatives to cultivate the passion for agriculture, rural areas, and rural residents of the talents in microbiology. A special curriculum was developed combining production, education, and research. The concept of quality education in five aspects was introduced to innovate the mode of farming-and-reading education. In recent years, these measures improved the students' knowledge in agriculture, invigorated their passion for agriculture, and fostered their skills for developing agriculture in Huazhong Agricultural University.

Abstract:Fermentation Process Design is a distinctive practical course offered by the College of Biology at Hunan University for the undergraduates majoring in biotechnology. This course aims to enhance students' practical skills and application abilities, encouraging them to apply their acquired knowledge of microbiology and fermentation engineering in practical settings such as fermentation factories. The traditional teaching of this course primarily relies on classroom lectures. However, due to the practical nature of topics such as operating fermentation production lines and designing fermentation equipment parameters, we found that simple classroom lectures were not effective in triggering students' enthusiasm and providing them with a clear understanding of the teaching content. Therefore, we reformed the teaching for this course by introducing the teaching model of "classroom+virtual simulation+factory", aiming to improve students' understanding of the knowledge about fermentation engineering and their practical and application abilities. This article introduces the current three-dimensional integrated model of "classroom+virtual simulation+factory" in the teaching of Fermentation Process Design and evaluates the teaching effectiveness with the feedback from students. We hope that our work will provide reference information for the teaching reform of colleges and universities.

Abstract:Microbiology Experiment is a foundation course for life sciences, food sciences, and other disciplines in colleges and universities. With strong practicality and applicability, this course plays an important role in the cultivation of innovative talents in biology. The current teaching of Microbiology Experiment in local universities face problems such as cramming teaching, dull content, low participation of students, and unreasonable assessment methods. Considering these problems and the requirements of engineering certification and first-class course initiative, we established a "123+2n" system for teaching reform and innovation. This system includes innovative reform of the teaching concept, teaching content, teaching methods, assessment methods, and evaluation methods. It aims to stimulate students' enthusiasm for learning, unleash their subjective initiative, and improve their innovation awareness and engineering practice ability, achieving the teaching goals of value shaping, knowledge impartment, and competence training. The results showed that this system significantly improved students' interest in learning, problem-solving abilities, and innovation abilities. This new system can provide reference for the teaching reform of experimental courses in local universities.