Abstract:[Background] Proteins produced by yeast usually undergo hyper glycosylation and form high mannose-type N-glycans, which can induce immune reactions in humans. As a result, the use of yeast is limited in the production of glycoprotein in drugs. Modifying the glycosylation pathway in yeast is a feasible approach for the production of glycoproteins used in drugs. Pichia kudriavzevii, a non-typical yeast species with strong stress resistance and rapid growth, has attracted much attention in recent years. By humanized glycosylation, P. kudriavzevii will serve as a cell factory to produce glycoproteins. [Objective] To obtain Man₅GlcNAc₂, the core structure of human N-glycan, is the key step to modify the glycosylation pathway in yeast. To achieve this goal, we deleted och1 of P. kudriavzevii and introduced msdS of Aspergillus into P. kudriavzevii. [Methods] The CRISPR-Cas9 system was employed to modify the N-glycosylation pathway of P. kudriavzevii, and the modified strain Δura3Δoch1::msdS was obtained. Furthermore, the N-glycocome of the glycoproteins secreted by Δura3Δoch1::msdS was analyzed. [Results] The N-linked Man₅GlcNAc₂ was detected on the glycoproteins secreted by Δura3Δoch1::msdS. [Conclusion] After the deletion of och1 and the introduction of msdS from Aspergillus,P. kudriavzevii cells can secrete the proteins with N-linked Man₅GlcNAc₂, which underpins the further humanized glycosylation in P. kudriavzevii.

Abstract:[Background] The pollution and accumulation of antibiotic resistance genes (ARGs) in coastal waters directly affect the quality and safety of seafood. As one of the four major fishing grounds, Haizhou Bay is the main carrier of fishery development in Jiangsu province, with multiple rivers injected. The coast is an important agricultural area with a significant impact on public health. [Objective] To study the microorganisms and ARGs in the water and sediment samples collected from six points of Haizhou Bay in summer and autumn. [Methods] Metagenomic sequencing was employed to determine the composition and relative abundance of microorganisms and ARGs. [Results] Proteobacteria and Actinobacteria were the most dominant phyla in the two media in summer and autumn. The dominant families in water and sediment samples were Rhodobacteraceae and Desulfobacteraceae, respectively. The relative abundance of ARGs in water samples in summer was significantly higher than that in autumn, while the relative abundance of ARGs in sediments did not show a significant trend in different seasons. The resistance mechanisms of the main phyla in water samples were mainly antibiotic target replacement and antibiotic target protection, while that in the sediment samples was mainly antibiotic inactivation. The main families had more diverse resistance mechanisms. Redundancy analysis (RDA) and canonical correspondence analysis (CCA) showed that environmental factors such as nitrate and salinity were significantly correlated with the changes of ARGs.[Conclusion] The microbial community and ARGs in the coastal area of Haizhou Bay varied in different media in summer and autumn, and the main microbial communities in different media adopt different resistance mechanisms. The relative abundance and distribution of ARGs were regulated by environmental factors such as salinity and nitrate. The findings provide a theoretical basis for public safety monitoring and environment protection in Haizhou Bay.

Abstract:[Background] Wetlands harbobring a variety of methane-producing archaea emit ample methane. The uncultured methanogens, Rice Cluster II (RCII), exist widely in cold acidic peatlands, freshwater lake wetlands, minerotrophic fens, and northern peat moss. However, the distribution and metabolic pathways of RCII in the low-temperature saline-alkaline wetlands remain unknown. [Objective] To investigate the diversity and infer the methanogenic pathways and potential adaptation mechanisms of RCII in saline-alkaline Zhalong wetland. [Methods] High-throughput sequencing of the 16S rRNA genes in the top layer of soil was performed to reveal the composition of methanogenic archaea. We established the 16S rRNA gene clone library to analyze the phylogenetic clusters of RCII. We then conducted metagenomic analysis to infer the methanogenic pathways and the possible saline-alkaline adaption mechanisms of the RCII in Zhalong wetland.[Results] The uncultured RCII methanogens accounted for 13.280%±0.019% of the total methanogenic archaea in Zhalong wetland and were clustered into three phylogenetic groups. Two metagenome-assembled genomes of RCII possessed the complete suite of genes involved in methanogenesis via reduction of carbon dioxide. Furthermore, the two RCII genomes contained the genes associated with the transport and synthesis of trehalose. [Conclusion] Abundant uncultured RCII methanogens inhabit the saline-alkaline Zhalong wetland, and they can produce methane by reducing carbon dioxide and use the compatible solute trehalose to adapt to the saline-alkaline environment.

Abstract:[Background] Methane-oxidizing bacteria play a role in maintaining the carbon balance of wetland ecosystems. Qinghai Lake alpine wetland has an important ecological status, while the methane-oxidizing bacteria in this area remain to be studied. [Objective] To explore the community characteristics and driving factors of methane-oxidizing bacteria in different types of alpine wetlands. [Methods] The methane-oxidizing bacteria in Xiaobo Lake swamp wetland, Bird Island lakeside wetland, and Wayanshan river source wetland were detected by high-throughput sequencing. [Results] The dominant phylum of methane-oxidizing bacteria in the three alpine wetlands was Proteobacteria. The alpha diversity of methane-oxidizing bacteria was different between Bird Island lakeside wetland and Wayanshan river source wetland (P<0.05), while it was similar between Xiaobo Lake swamp wetland and other two wetlands (P>0.05). The linear discriminant analysis effect size (LEfSe) revealed 40 differential microbial taxa between different types of alpine wetlands, and Wayanshan river source wetland had the most differential microbial taxa from the phylum to genus level. The redundant analysis showed that the main factors influencing the community structure of methane-oxidizing bacteria were soil temperature, soil moisture, and electrical conductivity. [Conclusion] The soil physical and chemical properties, methane-oxidizing bacterial diversity, and the relative abundance of several bacterial taxa (P<0.05) have differences among the three alpine wetlands around Qinghai Lake.

Abstract:Background China has abundant waste straw. The incomplete transformation of straw by microbial fermentation causes secondary pollution and resource wastes. The wastes from straw fermentation can be carbonized to prepare the adsorbent for removing organic pollutants from water. Objective To investigate the pollutant removal efficiency of biochar prepared from fermented straw, clarify the effects of fermentation process on the properties of straw biochar, and finally provide a theoretical basis for reusing the wastes from straw fermentation and removing organic pollutants from water. Methods Solid-state fermentation of wheat straw and rice straw was carried out with Trichoderma reesei QM6a and T. asperellum T-1, respectively. The fermentation residues were then pyrolyzed to adsorb methylene blue (MB). Results The fermentation of straw effectively shortened the time to adsorption equilibrium and improved the MB adsorption efficiency of wheat straw biochar. The adsorption efficiency of MB (50 mg/L) by the biochar pyrolyzed at 600 ℃ from wheat straw pre-fermented by T. asperellum T-1 (BaWS 600) was 53.6% higher than that from natural straw (BWS 600). The pseudo-second-order model could well fit the adsorption process, and the MB adsorption capacity of BaWS 600 was 119.4% and 299.4% higher than that of BWS 600 and BWS 800, respectively. Freundlich isothermal adsorption equation could well characterize the isothermal adsorption process, which indicated the multi-molecular layer adsorption of MB by straw biochar. Straw fermentation increased the specific surface area of wheat straw biochar by 47.4% to 245.8% and promoted the exposure of oxygen-containing functional groups, which might be the main reasons for the improvement of MB adsorption efficiency. Conclusion Fermentation can effectively improve the properties of straw biochar for pollutant removal. T. asperellum T-1 was identified to be more suitable for straw fermentation to improve the pollutant adsorption performance of biochar because of its stronger cellulase-secreting ability.

Abstract:[Background] Poa pratensis is a species of widely used ornamental and facility cold-season turfgrass. Powdery mildew caused by Blumeria graminis f. sp. poae seriously reduces the lawn quality and shortens the service life of P. pratensis.[Objective] To isolate, identify, and determine the pathogenicity of the isolate BGP(TG) and provide a theoretical foundation for the prevention and control of powdery mildew in P. pratensis. [Methods] BGP(TG) isolated from a greenhouse of Shanxi Agricultural University was identified based on the morphological characteristics and ribosomal DNA internally transcribed spacer (rDNA-ITS) sequence. The pathogenicity of the isolate was determined with 23 samples of P. pratensis.[Results] The conidiospores of the pathogen were in chains, colorless, and oval, with the size of (23.31−34.67) µm×(11.33−15.77) µm and finger-like mature haustoria. The phylogenetic analysis based on rDNA-ITS sequences showed that the BGP(TG) isolate and B. graminis f. sp. poae shared the same branch. The pathogenicity tests showed that the disease index ranged from 14.41 to 53.12, and the incidence ranged from 49.47% to 77.72% nine days after 23 samples of P. pratensis were inoculated with BGP(TG). Only the native species ‘Taihang’ in Shanxi Province showed moderate resistance to BGP(TG).‘Yingxian’, ‘Black Jack’, and ‘Hunyuan’ showed moderate sensitivity, and ‘Explorer’ and ‘Award’ showed extreme sensitivity. The other 17 species showed high sensitivity, accounting for 73.91%. [Conclusion] The pathogenicity of BGP(TG) in Shanxi Province was strong. Mining the genes conferring resistance to powdery mildew from P. pratensis ‘Taihang’ will be of great significance for the breeding of P. pratensis with resistance to powdery mildew.

Abstract:[Background] Ganoderma lucidum is a precious Chinese medicinal herb with both medicinal and edible values. Adenosine, one of the active components, endows the herb with immunomodulatory, anti-inflammatory, and anti-tumor activities. [Objective] To regulate the expression of key genes involved in adenosine biosynthesis to increase the adenosine production of G. lucidum. [Methods] We studied the correlations of the expression of adenosine synthase genes (GlATIC,GlPNP, and GlADK) with the adenosine content. The key enzyme genes that were positively correlated with the adenosine content of G. lucidum were screened out. The key enzyme genes in G. lucidum were cloned and overexpressed to investigate the effect of key enzyme gene overexpression on adenosine accumulation. [Results] The expression of GlPNP was positively correlated with adenosine content. The key gene GlPNP was cloned and overexpressed in G. lucidum. The cDNA of GlPNP was 969 bp in length. GlPNP displayed a trimeric quaternary structure, with a predicted molecular weight of 34.6 kDa. The expression levels of GlPNP in the transformants were 2.9–3.9 folds higher than that of the wild type on day 4. The adenosine content in the transformants increased by 78% and 63%, respectively, compared with that in the strain transformed with the blank vector. [Conclusion] Overexpression of GlPNP is an effective strategy to increase the production of adenosine.

Abstract:[Background] As a special structure of plant-microorganism symbiosis, the root nodules of legumes contain abundant microbial resources. The siderophore-producing endophytic bacteria in the root nodules can promote nodulation and nitrogen fixation. However, it remains unclear whether these bacteria have a direct growth-promoting effect on plants. [Objective] To screen out, identify, and examine the growth-promoting effect of the siderophore-producing endophytic bacteria in the root nodules of Lespedeza daurica, so as to provide a new way for enriching plant growth-promoting bacterial resources. [Methods] The CAS medium was used for the preliminary screening of efficient siderophore-producing endophytic bacteria in the root nodules. The liquid fermentation was carried out to examine the siderophore production. The growth-promoting functional media were used to measure the growth-promoting effects of the strains. The stress resistance of the strains was determined with the YMA liquid medium with different salt concentrations (0.01%–9.00%), pH 3.0–12.0, and culture temperatures (4–60 ℃). The pot experiment was carried out to preliminarily verify the direct growth-promoting effect. [Results] Forty-eight strains possessed siderophore-producing ability, including 9 efficient strains, which were TG4, TG9, TG13, TG15, TG28, TG32, TG38, TG76, and TG78, respectively. The siderophore production of the strains varied within 26%–93%. Except that TG28 was identified as Priestia megaterium, the other 8 strains were identified as Brevibacterium frigoritolerans. TG15, TG28, TG76, and TG78 could secrete indole-3-acetic acid (7.62–64.90 μg/mL) and ACC deaminase, fix nitrogen, and grow at 0.01%–5.00% NaCl, pH 4.0–12.0, and 4–37 ℃. TG15 and TG28 significantly increased the stem diameter, and TG28 significantly increased the plant height of L. daurica.[Conclusion] There were siderophore-producing bacteria with growth-promoting effects and stress resistance in the root nodules of L. davurica. However, the growth-promoting effect on plants was only reflected on the morphological development. The results indicate that the growth-promoting substances such as siderophores may have a positive effect on the nitrogen-fixing bacteria.

Abstract:[Background] The rhizosphere soil harbors diverse microorganisms. The isolation and screening of efficient strains with different functions is the premise for the development of efficient composite microbial fertilizers. [Objective] To isolate microorganisms from the rhizosphere soil and root surface of peanut and screen out efficient strains with organic and inorganic phosphorus-solubilizing, indole-3-acetic acid (IAA)-producing, and siderophore-producing functions, so as to lay a foundation for the research and development of microbial fertilizers for peanut. [Methods] The dilution-plate coating method was employed to isolate the microorganisms from the peanut rhizosphere soil and root surface samples collected from Qixia City, Pingdu City, and Laishan district of Yantai City in Shandong Province. The microorganisms were identified by the phylogenetic analysis based on the 16S rRNA gene sequences. The strains with efficient organic and inorganic phosphorus-solubilizing, IAA-producing, and siderophore-producing functions were obtained through primary and secondary screening. [Results] A total of 147 strains were isolated, purified, and preserved in this study, including 75 strains isolated from the rhizosphere soil samples and 72 strains from the root surface samples. The isolates belonged to 40 genera of 4 phyla including Actinomycetota, Bacillota, Bacteroidota, and Pseudomonadota. Streptomyces (21.77%) and Bacillus (16.33%) were the dominant genera. The root surface samples had higher alpha diversity of microbial community than the rhizosphere samples. Among the 62 strains with organic phosphorus-solubilizing ability, Brevundimonas sp. YTU21021 showed the highest activity of 1.12 mg/L. Among the 31 strains with inorganic phosphorus-solubilizing ability, Acinetobacter sp. YTU21009 showed the highest activity of 7.04 mg/L. Among the 63 strains that could produce IAA, Enterobacter sp. YTU21054 showed the highest yield of 184.19 mg/L. Among the 7 strains capable of producing siderophores, Burkholderia sp. YTU21051 presented the highest efficiency with the A_s/A_r ratio of 0.9. [Conclusion] There are diverse culturable microorganisms in the rhizosphere and root surface samples of peanut. The efficient functional strains obtained in this study enrich the functional microbial resources in peanut rhizosphere and lay a foundation for the development of composite microbial fertilizers with efficient rhizobia for peanut.

Abstract:[Background] Chitosan (CTS) is a polysaccharide ubiquitous in crustaceans. CTS has broad-spectrum antifungal activities, while whether CTS affects the growth and development of Aspergillus carbonarius and Fusarium sulphureum remains unknown. [Objective] To determine the effects of different concentrations of CTS on the growth and development of A. carbonarius and F. sulphureum. [Methods] The colony diameters, biomass, and mycelial dry weights of the two fungal species cultured in the potato dextrose agar (PDA) media with different concentrations of CTS were determined. The inhibitory effects of CTS on the two fungal species were evaluated by comparison of the sporulation, spore germination, and germ tube length. [Results] CTS treatment significantly altered the colony morphology of both fungal species. Higher concentrations of CTS resulted in more obvious colony shrinkage and deformation. CTS reduced colony diameter, mycelial dry weight, and mycelial biomass of both fungal species in a concentration-dependent manner, with stronger inhibitory effect on F. sulphureum. Similarly, CTS inhibited the sporulation, spore germination, and germ tube elongation in a concentration-dependent manner, which was more significant on F. sulphureum. The EC₅₀ values of CTS on A. carbonarius and F. sulphureum were 0.12 mg/mL and 0.075 mg/mL, respectively. [Conclusion] CTS inhibited the growth and development of A. carbonarius and F. sulphureum in a concentration-dependent manner, with stronger effect on F. sulphureum.

Abstract:[Background] Substrate utilization is an important physiological process in the cultivation of Sparassis latifolia, while little is known about the metabolite differences in the substrates at different developmental stages.[Objective] To identify the differential metabolites in the cultivation substrates of S. latifolia at different developmental stages, explore key metabolites, and provide a theoretical basis for the research on the mechanism of substrate utilization. [Methods] Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was employed to determine the changes of metabolites in the cultivation substrates of mycelium (Myc), primordium (Pri), and fruiting body (FB). Functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were conducted for the metabolites. The content of phytohormones in S. latifolia was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). [Results] A total of 1 360 metabolites were identified from the substrates of S. latifolia at three different stages. There were 179 common differential metabolites among three different comparison groups (Pri vs. Myc, FB vs. Myc, and FB vs. Pri), and the top 50 most abundant metabolites were mainly amino acids, lipids, pyranic acids, pyranones, and phytohormones. The content of amino acids gradually decreased while that of pyranic acids and pyranones gradually increased with the development of S. latifolia. The content of gibberellins (GAs) was higher in the substrates of Pri and FB, and that of jasmonic acid was higher in the substrate of Myc. GA7 was detected only in Pri, and the content of 12-oxo-phytodienoic acid was higher in FB. The differential metabolites were mainly enriched in purine metabolism, sphingolipid, glycerophospholipid metabolism pathways. [Conclusion] Amino acids are key metabolites in the substrate of Myc. pyranoic acids, pyranones, and phytohormones may be involved in the physiological and biochemical regulation of substrate utilization in the Pri and FB stages.

Abstract:[Background] Plants can mitigate stress by modulating the structure of rhizosphere microbial community, while the changes of wheat growth and rhizobacterial community structure under saline-alkaline stress remain to be clarified. [Objective] To characterize the growth and rhizobacterial community structure of wheat seedlings under saline-alkaline stress. [Methods] Saline-alkaline stress was simulated via the adjustment of the Na⁺ concentration and pH of Hoagland nutrient solution with NaCl and Na₂CO₃, and MiSeq high-throughput sequencing was employed to determine the bacterial community structure. [Results] Compared with no-stress treatment, the saline stress alone decreased the aboveground dry weight, belowground dry weight, functional leaf chlorophyll content, and available nitrogen in rhizosphere soil by 25.0%, 57.1%, 21.2%, and 42.9%, respectively (P<0.05). The increase in pH reduced plant height, root length, biomass, functional leaf chlorophyll content, and the content of main nutrients (available nitrogen, available phosphorus, and organic matter) and the activities of enzymes (alkaline protease, alkaline phosphatase, sucrase, urease, and dehydrogenase) in rhizosphere soil (P<0.05). Three phyla (Proteobacteria,Actinobacteria, and Firmicutes with the relative abundance of 7.1%–71.5%) and five genera (Enterobacter, Methyloversatilis, Azotobacter,Pseudomonas, and Allorhizobium with the relative abundance of 0.5%–5.1%) in the rhizosphere of wheat seedlings were dominant in all the treatments. Saline-alkaline stress had no obvious effect on the alpha diversity indexes, including Shannon index and Chao1, while it significantly affected the beta diversity and changed the community structure. The saline stress alone increased the relative abundance of Flavobacterium, Pseudomonas, Methyloversatilis,Azotobacter, and some unclassified genera by 0.27%–3.12%. The increase in pH increased the relative abundance of Enterobacter, Noviherbaspirillum, and Bacteroidetes. [Conclusion] Saline-alkaline stress significantly inhibited the growth of wheat seedlings and remarkably changed the rhizobacterial community structure. The increased relative abundance of bacterial taxa such as Enterobacter in response to saline-alkaline stress may promote the growth of wheat seedlings and be major microbial sources for improving the crop productivity in saline fields.

Abstract:[Background] Southern blight is a soil-borne disease caused by Sclerotium rolfsii, causing serve loss to Aconitum carmichaelii production. [Objective] To clarify the taxonomic status of an antagonistic strain CZ1 against S. rolfsii, and optimize the fermentation conditions to maximize the control effect on southern blight of A. carmichaelii. [Methods] The dilution-plate coating method was employed to isolate bacteria from the Malpighian tubules of Cantheconidae furcellata. The plate confrontation assay was employed to screen out the strain with strong resistance to S. rolfsii, and the inhibitory effects of this strain on other plant pathogenic fungi were determined. The taxonomic status of the strain was determined based on morphological characteristics, physiological and biochemical properties, and 16S rRNA gene sequence. With OD₆₀₀ value of the fermentation broth as the indicator, single factor and orthogonal experiments were carried out to optimize the medium components and fermentation conditions. Furthermore, indoor pot experiment was carried out to evaluate the control effect of the strain. [Results] Six strains of bacteria were isolated, among which CZ1 had the highest inhibition rate on S. rolfsii. CZ1 had inhibitory effects on other plant pathogenic fungi such as Phytophthora nicotiana and Fusarium oxysporum and was preliminary identified as Bacillus subtilis. The optimal culture medium was composed of 15.00 g/L sucrose, 25.00 g/L peptone, and 7.50 g/L yeast extract. The strain showed the highest biomass when cultured at the inoculation amount of 2%, liquid loading volume of 90 mL, 210 r/min, 36 ℃, and initial pH 8.0 for 120 h. The results of indoor pot experiment showed that the average control efficiency of CZ1 against the southern blight on A. carmichaelii reached 59.26%. [Conclusion] CZ1 strain has good potential in the biocontrol of southern blight on A. carmichaelii.

Abstract:[Background] Rice blast, one of three major diseases of rice, causes great losses to rice production every year. Biocontrol has become a key means for the prevention and control of this disease. However, there are few endophytes isolated from Oryza officinalis Wall. for the control of this disease at present. [Objective] To excavate the microbial resources against rice blast from the endophytes of O. officinalis. [Methods] The endophytes were isolated from O. officinalis, screened by the plate confrontation method, and identified based on morphological characteristics and 16S rRNA gene sequences. Furthermore, we optimized the medium, pH, temperature, and shaking speed for the fermentation of the strain screened out. [Results] The antagonistic strain Z5 was screened out and identified as Bacillus pumilus, with the inhibition rate of 96.43% on the mycelial growth of Magnaporthe grisea in the LB medium. Strain Z5 showed the best growth in the NYBD medium among the five medium tested (NYBD, CM, LB, NA, and YSP) and at pH 8.0–9.0, 35 ℃, and 250 r/min. [Conclusion] B. pumilus Z5 exerted a good inhibitory effect on the growth of M. grisea and can tolerate high temperature, acid, and alkali, demonstrating the potential of serving as a biocontrol agent for rice blast.

Abstract:[Background] Cymbidium ringspot virus (CymRSV) is an important phytosanitary virus, which is seriously harmful in the world. [Objective] To establish a specific and sensitive assay for the quantitative analysis of CymRSV. [Methods] According to the highly conserved region in five coat protein gene sequences of CymRSV, we designed three pairs of primers and three probes, and then obtained the target sequence (145 bp) and screened out the optimal primers and probe. We then used the target sequence as a template to construct a positive recombinant plasmid and established a standard curve, on the basis of which the sensitivity, specificity, stability, and performance of the established assay were explored. [Results] In this study, the established RT-qPCR detection method for CymRSV showed that the minimum limit of detection of 1 copy for the positive plasmid standards and the minimum limit of stable detection of 5 copies/μL, demonstrating the sensitivity 10³–10⁴ times that of RT-PCR. The standard curve was y=−3.332x+40.371 (R²=0.999), which showed a good linear relationship between C_t value and logarithm of copy number and the amplification efficiency was 99.6%. The assay showed good specificity as it generated no amplification curve for other 5 common viruses. The intra-group and inter-group coefficients of variation of C_t value were less than 0.6%, which suggested good repeatability and stability. Furthermore, the established assay was employed to detect 20 samples of 4 orchid species. The positive control showed an amplification curve at C_t of 23.31, and the negative control and the samples showed no amplification curves. [Conclusion] The successful establishment of CymRSV RT-qPCR detection method based on TaqMan probe can provide technical support for the accurate detection and scientific prevention and control of orchid CymRSV virus.

Abstract:[Background] Stropharia rugosoannulata is a new edible mushroom species in China. With simple cultivation methods and high yield and benefits, it plays a key role in the efficient transformation of agricultural wastes and the increase in farmers’ income. [Objective] To explore the effects of selenium on the mycelial growth, agronomic traits, and nutritional quality of S. rugosoannulata and provide a basis for the production of selenium-enriched S. rugosoannulata. [Methods] The mycelial growth rate and dry weight of S. rugosoannulata treated with different concentrations of sodium selenite (Na₂SeO₃) were determined by the plate culture method. The cultivation matrix composed of sawdust, rice husk, and corncob was supplemented with 0–10 mg/kg Na₂SeO₃ for the cultivation test. The agronomic traits, nutritional quality, and antioxidant activity of the first batch of fruiting bodies were evaluated. [Results] The treatments with 0–10 mg/L Na₂SeO₃ had no significant effects on the mycelial growth rate and dry weight, which were 3.74–3.76 mm/d and 40.67–41.33 mg, respectively. However, when the concentration of Na₂SeO₃ reached 15 mg/L or above, the mycelial growth was significantly inhibited. The cultivation tests showed that the addition of 0–10 mg/kg Na₂SeO₃ had no significant effects on the biological efficiency of the mushroom. The 7.5 mg/kg Na₂SeO₃ group showed the best cap diameter, stipe diameter, and fresh weight of single mushroom. Moreover, this group demonstrated the highest content of crude protein, crude polysaccharides, total amino acids, essential amino acids, non-essential amino acids, delicious amino acids, and glutamate. The crude polysaccharides from fruiting bodies in the 7.5–10.0 mg/kg Na₂SeO₃ groups demonstrated significantly higher DPPH and · OH free radical scavenging activities than the blank control group. [Conclusion] The addition of low-dose Na₂SeO₃ could significantly improve the agronomic traits and nutritional quality of the fruiting bodies of S. rugosoannulata, and the optimum dose was 7.5 mg/kg. The findings provide a reliable experimental basis for the production of selenium-enriched S. rugosoannulata.

Abstract:[Background] Clostridium butyricum is a new generation of obligate anaerobic spore-producing probiotics with strong tolerance to heat, acid, and stress, demonstrating a great application value and development prospect. [Objective] To optimize the fermentation medium of C. butyricum and study the activities of the fermentation broth in terms of inhibiting Aspergillus flavus and degrading aflatoxin B₁ (AFB₁). [Methods] Response surface methodology was employed to optimize the fermentation medium. The oxford cup assay was employed to examine the inhibitory activity of C. butyricum fermentation broth on A. flavus and enzyme-linked immunosorbent assay to determine the AFB₁-degrading ability of the fermentation broth. [Results] The optimized fermentation medium was composed of 18.1 g/L glucose, 29.7 g/L soya peptone, 3.8 g/L K₂HPO₄· 3H₂O, 2.0 g/L NaCl, 4.0 g/L NaAc, 1.2 g/L MgSO₄· 7H₂O, and 0.3 g/L l-cystine hydrochloride. After optimization, the biomass of C. butyricum reached 2.28×10⁹ cells/mL, which was 2.54 times of that (8.99×10⁸ cells/mL) before optimization. The fermentation broth of C. butyricum exerted a significant inhibitory effect on A. flavus, and the concentrated supernatant degraded 68.65% of AFB₁ within 72 h. The results indicated that the active components degrading AFB₁ in the supernatant were the extracellular enzymes produced by C. butyricum.[Conclusion] The biomass of C. butyricum was significantly increased by fermentation medium optimization, and the fermentation broth of C. butyricum inhibited A. flavus and degraded AFB₁. The findings provide scientific evidence for the industrial production of C. butyricum and the development and application of microecological preparations.

Abstract:[Background] The bactericidal hypochlorous acid water may negatively affect the beneficial bacteria in the intestinal tracts of domesticated animals when being used as drinking water. [Objective] To isolate the lactic acid bacteria with tolerance to slightly acidic hypochlorous acid water (SAHW) and investigate their probiotic properties. [Methods] Eight strains of Lactobacillus were isolated from the traditional natural fermentation products in Inner Mongolia and identified by 16S rRNA gene sequencing. The tolerance to SAHW, acid, and bile salt, hydrophobicity, and self-agglutination properties of the isolates were examined. [Results] The strain MBH3-2 was identified as Lactobacillus paracasei. After treatment with 20 mg/L SAHW for 2 h, the strain showed the count decrease of 0.3 lg(CFU/mL) and the survival rate of 50.10%. MBH3-2 was highly hydrophobic with the hydrophobicity of 62.12%, and its self-agglutination rate reached 97.29% at the time point of 2 h. [Conclusion] Strain MBH3-2 with strong tolerance to SAHW and basically meeting the requirements as a microecological preparation was identified, demonstrating the potential to be developed as a new microecological preparation.

Abstract:[Background] Riemerella anatipestifer (RA), a major pathogen that causes losses in the poultry industry, can cause serositis and septicemia in ducks and other birds. The available vaccines generally have weak reaction and low immunogenicity and induce low antibody levels. [Objective] To explore the immune response and protective effect of CpG adjuvant and provide evidence for the prevention of duck serositis with CpG adjuvant. [Methods] The proliferation test of lymphocytes in duck peripheral blood and real-time fluorescence quantitative PCR were employed to determine the optimal sequence. The recombinant plasmid pUC18-CpG was constructed and compared with CpG ODN for immunogenicity. Ducklings were immunized via subcutaneous injection twice with inactivated RA-GH5 as the antigen and pUC18-CpG as the adjuvant, and then the serum antibody titer and cytokine level were determined. The ducklings were then challenged by RA-GH5 strain through intramuscular injection, and the histopathological changes and immune protection rate were examined. [Results] The optimal sequence CpG-3 was screened out. The immunostimulatory activity of pUC18-CpG containing CpG-3 was not significantly different from that of CpG-3. Compared with RA vaccine alone, the addition of pUC18-CpG significantly increased the serum antibody titer and the levels of interleukin-2 (IL-2) and IL-4. The immune protection rates of 80 μg, 20 μg, and 40 μg pUC18-CpG to the ducklings were 70%, 60%, and 60%, respectively. [Conclusion] The combination of pUC18-CpG and RA-GH5 inactivated vaccine can induce stronger humoral and cellular immune responses.

Abstract:Background Cannabinoids are natural active products of plants that serve as key clinical drugs. At present, the production of pharmaceutical cannabinoids still relies on plants, which has low yield, long cycles, and safety problems. Biosynthesis is of great significance to the production of cannabinoids and their analogues. Objective To reconstruct the biosynthetic pathway of the cannabinoid precursor sphaerophorolcarboxylic acid (SA) in Saccharomyces cerevisiae BJ5464-npgA and optimize the fermentation medium to increase the yield of SA. Methods Mr_OvaA, Mr_OvaB, and Mr_OvaC were amplified by PCR with the genomic DNA of Matarhizium robertsii ARSEF 23 as the template and respectively integrated into S. cerevisiae-Escherichia coli shuttle plasmids. All the three recombinant plasmids were transformed into S. cerevisiae BJ5464-npgA and the engineered strain CLB2 was obtained. Single factor experiments, Plackett-Burman design, method of steepest ascent, and response surface method were employed to optimize the fermentation medium. The experimental data were analyzed in Design Expert 8.0. Results The S. cerevisiae engineered strain CLB2 yielded 63.75 mg/L SA. The three main factors affecting SA yield were sucrose, KH₂PO₄, and vitamin solution. The optimized medium was composed of 7.26 g/L sucrose, 6.08 g/L KH₂PO₄·7H₂O, 5.67 mL/L vitamin solution, in which the predicted SA yield was 93.15 mg/L. The actual yield of SA in the optimized medium was 93.75 mg/L, which was 47% higher than that before optimization. Conclusion Cannabinoid precursor SA can be synthesized efficiently in S. cerevisiae. The fermentation medium formula for high production of SA in this study provides reliable support for the subsequent research on SA and cannabinoid.

Abstract:[Background] Phlebopus portentosus is the first edible species of Boletales that can be cultivated artificially, and the number of sclerotia varies during the artificial cultivation of different strains. [Objective] To explore the different mechanisms of sclerotium production of different strains. [Methods] The mature sclerotia of the strain with multiple sclerotia (JH1) and the strain with oligo-sclerotia (JH2) and the mycelia of the strain with no sclerotium (JH3) cultivated for the same time period were collected. Transcriptome sequencing was performed to identify the differentially expressed genes (DEGs) in sclerotium formation, and the roles of these DEGs were analyzed. [Results] The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment showed that the DEGs between JH2 and JH1 were mainly enriched in the phenylalanine, tyrosine, and tryptophan biosynthesis, arginine, proline metabolism, cysteine and methionine metabolism. The DEGs between JH3 and JH1 were mainly involved in glyoxalate and dicarboxylate metabolism, and those between JH3 and JH2 in glutathione metabolism and glyoxylate and dicarboxylate metabolism. Among the sclerotium formation-associated DEGs between JH2 and JH1, between JH3 and JH1, and between JH3 and JH2, respectively, 69, 118, and 82 genes were associated with signal transduction, sensory stimulation, defense, and carbohydrate active enzymes, and those associated with carbohydrate active enzymes had the highest abundance. The expression of carbohydrate active enzyme genes shared by the three comparison groups in JH1 was higher than that in JH2 and JH3, indicating that JH1 could make full use of the substrate to form more sclerotia. [Conclusion] The differences in sclerotium production among different strains of P. portentosus were preliminarily analyzed at the transcriptome level, which provided a theoretical basis for the subsequent research on the correlations of sclerotia with fruiting and yield.

Abstract:Soil cadmium pollution seriously endangers the safe production of agricultural products, while plant rhizosphere bacteria play a critical role in passivating cadmium in soil and helping crops resist cadmium stress. This paper firstly summarizes the species of rhizosphere bacteria used widely in the remediation of cadmium-contaminated soils and elaborates on the mechanisms of cadmium passivation by plant rhizosphere bacteria from four aspects: direct adsorption of cadmium by rhizosphere bacteria, adjustment of soil physical and chemical characteristics, regulation of rhizosphere bacterial community and other effects. Secondly, from the perspective of bacterium-plant interaction, we expound the effects of the interactions between plant root exudates and rhizosphere bacterial community changes on soil cadmium passivation. Finally, we prospect the core flora assembly of plant rhizosphere bacteria for passivating cadmium under heavy metal stress. With this review, we aim to explore the molecular mechanism of plant root-microorganism interaction system in the context of emerging disciplines and advancing technologies and facilitate the in-depth theoretical research and practice on the remediation of heavy metal-contaminated soil by rhizosphere bacterial passivation.

Abstract:Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has rapidly spread around the globe since early 2020, becoming one of the worst pandemics in the history. The spike (S) protein of SARS-CoV-2 can bind to angiotensin-converting enzyme 2 (ACE2), a receptor on the host cell surface, to cause gut microbiota imbalance and different complications. Probiotics, proved to be capable of regulating gut microbiota to treat diseases and viral infections, have been considered to serve as an option for alleviating COVID-19. We summarize the efficacy of probiotics in alleviating clinical symptoms and gastrointestinal adverse events of COVID-19 based on the results of available preclinical and clinical trials. Furthermore, we discuss the potential of probiotics to mitigate the sequelae of COVID-19, thereby providing new directions for the subsequent management of COVID-19 and a theoretical basis for treating respiratory diseases.

Abstract:Quorum sensing inhibitors target the quorum sensing system ubiquitous in pathogenic bacteria to block the biofilm formation or regulate the expression of virulence genes without threatening bacterial growth. Therefore, they are less likely to trigger bacterial resistance and serve as ideal antibacterial synergists. Molecular docking is one of the virtual screening techniques, characterized by a clear target, high efficiency, low cost means of drug research and development. This paper introduces the main modules of molecular docking and the advances in the application of this technique in the screening of quorum sensing inhibitors.

Abstract:Isoprenoids, a family of natural molecules with high chemical diversity are involved in the synthesis of secondary metabolites such as carotenoids and sterols in microorganisms, demonstrating a promising commercial prospect for industrial production. Prenyltransferase (PT), a key enzyme in the synthesis of isoprenoids, regulates the production of secondary metabolites. In this paper, we review the recent studies about the isolation and identification of prenyltransferases in microorganisms and introduce their structural features, chain length determination mechanisms, and complex evolution. Furthermore, we overview the applications of the regulation of enzyme gene expression and the current status of research on biosynthesis, aiming to provide ideas for in-depth study of the mechanism of action and applications of prenyltransferases.

Abstract:Compared with traditional techniques for microbial identification, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an accurate, reliable, and rapid technology for identification and typing. We reviewed the relevant research papers in recent years and summarized the latest research progress. MALDI-TOF MS has great advantages in the identification of clinical pathogenic microorganisms, foodborne microorganisms, and environmental microorganisms, which accelerates the process of microbial identification. Furthermore, we explored the latest progress and challenges of this technology in new fields, aiming to provide reference for the development of MALDI-TOF MS in China.

Abstract:Microbial cells are often subjected to acid stress in natural environments or industrial applications, which severely restricts cell growth and product synthesis efficiency. In order to survive in acidic environments, acid-resistant bacteria have developed diverse protective mechanisms such as hydrogen ion consumption, membrane protection, and metabolic modification to maintain intracellular pH homeostasis. Therefore, in-depth research on acid-resistant mechanisms and improving acid resistance of strains are important for microbial biosynthesis of value-added products. As a model microorganism, Escherichia coli has been well studied regarding the acid-resistant mechanisms. In recent years, significant progress has been achieved in the research on the acid-resistant modification of E. coli. This paper reviews the acid-resistant mechanisms of E. coli in terms of oxidative or glucose-repressed system (acid resistance system 1, AR1), glutamate-dependent acid resistant system (acid resistance system 2, AR2), arginine-dependent acid resistant system (acid resistance system 3, AR3), lysine-dependent acid resistant system (acid resistance system 4, AR4), ornithine-dependent acid resistant system (acid resistance system 5, AR5), cell membrane protection, and biomolecular repair. Furthermore, we summarize the progress in constructing acid-resistant E. coli strains by metabolic engineering, global transcriptional engineering, and adaptive laboratory evolution. Finally, we discuss the subsequent research directions for further deciphering the acid-resistant mechanisms and improving acid resistance of E. coli.

Abstract:Vibrios are ubiquitous in aquatic environments. The outbreaks of vibriosis cause substantial economic losses to the mariculture industry and seriously threaten human life and health. Some vibrios can improve their survival and environmental persistence and spread across the ocean through interactions with phytoplankton and zooplankton. We describe the role and influencing factors of marine plankton as reservoirs of some vibrios and the plankton-associated transmission characteristics of vibrios upon climate change and human activities. Focuses are put on the unique roles and mechanisms of the specific structures, metabolic pathways, and secondary metabolites of vibrios in the interactions with plankton. Further research should focus on the molecular mechanism of interactions between plankton and vibrios and the mechanism of plankton-associated spreading of vibrios on the global scale. This review provides meaningful information for preventing and controlling the risk of plankton-associated transmission of vibrios.

Abstract:Extracellular electron transfer (EET) refers to the process of transferring electrons generated in microorganisms to other electron acceptors to obtain energy, and the electron transfer that occurs between two different microorganisms is called microbial interspecies electron transfer (IET). IET can occur in two ways: mediated interspecies electron transfer (MIET) and direct interspecies electron transfer (DIET). Without of the need for other material mediation, DIET has higher efficiency and energy utilization efficiency than MIET. This paper systematically expounds the mechanisms and applications of EET in microorganisms, with focus on the mechanisms and potential application fields of DIET. This review aims to provide a reference for finding more electrically connected microbial communities and applied microorganisms.

Abstract:Staphylococcus aureus is a major contributor to the microbiological safety hazards in China. It uses fatty acids to synthesize phosphatidic acid (an essential intermediate in the synthesis of cell membrane phospholipids) for reproduction. Therefore, inhibiting the synthesis of phosphatidic acid can effectively control S. aureus and thus reduce the damage to the environment and organisms. However, S. aureus has the ability to synthesize phosphatidic acid via both the type II fatty acid synthesis (FASII) pathway and the FASII bypass. The common inhibitors against S. aureus only target the FASII pathway, which can result in the emergence of FASII bypass escape when the bacteria are exposed to high levels of exogenous fatty acids, creating a potential gap in the protection. This paper provides a comprehensive overview of the signaling processes of the FASII pathway and bypass for synthesizing phospholipid acid, as well as the conversion of phospholipid acid to other phospholipids in S. aureus. Furthermore, the key targets of the signaling processes that may be inhibited by antibacterial agents are discussed. This review may provide theoretical guidance for the development of new bacterial inhibitors.

Abstract:With the development of virtual simulation experiment teaching in China, group learning has attracted increasing attention and been applied to practical teaching. However, there are few studies about the teaching practices combining group learning with virtual simulation experiment courses. In view of the typical problems existing in the practical teaching of microbial experiments, we took the Microbial Physiology course for postgraduates at Harbin Institute of Technology as an example to illustrate the virtual simulation experiment teaching based on collaborative and interactive learning, aiming to create a first-class experiment/practice course of microbiology. This paper describes the connotation, elements, and advantages of collaborative and interactive learning, discusses the application of this learning mode in virtual simulation experiment teaching of Microbial Physiology, and summarizes the deficiency and development direction of this teaching method.

Abstract:[Background] Actinomycetes exhibit rich genetic and functional diversity and produce a wide range of secondary metabolites, which play a role in clinical medicine, agricultural production, and pollution control. Marine actinomycetes have attracted much attention due to their special metabolic pathways and their ability to produce unique active natural products. [Objective] To investigate the research hotspots and future trends of marine actinomycetes and provide reference for subsequent research. [Methods] The articles about marine actinomycetes were retrieved from Web of Science with marine actinomycetes or marine actinobacteria as the keyword for quantitative analysis. VOSviewer was used for the visual analysis of the keywords, countries, institutions, authors, and publishing dates. [Results] The total number of articles about marine actinomycetes was increasing year by year. China and the United States far exceeded other countries in the number of articles and the frequency of citations. The relevant studies focused on the isolation and identification of strains, the mining of active natural products, and bioinformatics. [Conclusion] Marine actinomycetes have attracted increasing attention worldwide. Domestic and overseas institutions should strengthen cooperation and employ bioinformatics to further mine the active secondary metabolites and facilitate the development of marine actinomycetes.