Abstract:[Background] When Escherichia coli K-12 mutant S17-3 carries high copy number plasmid pBHR68 expressing the gene cluster for synthesis of poly-3-hydroxybutyrate (PHB), it presents some special physiological features, such as high-density growth, low pH tolerance, and high yield of colonic acid (CA) at low pH. [Objective] To systematically explore the molecular mechanism of high-density growth that related to strain (E. coli S17-3) and plasmid (pBHR68), and to reveal the coupling mechanism between the high-density growth and the anabolism of PHB or CA. [Methods] We dissected the plasmid composition and the gene structure for CA synthesis pathway that might have caused high-density growth, searched for key mutations by multiple genome alignment analysis. The transcriptomic data of E. coli S17-3 and its growth performances in different media were scrutinized, the functions of the verified genes were analyzed by gene knockout. [Results] The high-density growth of E. coli S17-3 was related to the overexpression of the whole gene cluster for PHB synthesis, as well as the multi-site mutations within rhsA; RcsA may play as a key factor that not only regulates the production of CA but also mediates the channeling of carbon flow during E. coli S17-3's growth; The knockout of key enzymes in CA operon led to the increase of biomass at low pH; In addition, the high-density growth of E. coli S17-3/pBHR68 may also be related to lactose metabolism, since lacZ null mutation abolished the capability of CA synthesis as well as the high-density growth. [Conclusion] In this study, we analyzed the factors that may link to the high-density growth of E. coli S17-3, and found out some important clues which laid a research foundation for studies to remold E. coli S17-3 as a chassis cell for production of oligosaccharides.

Abstract:[Background] Ramie fiber is known as the "king of natural fiber" and used in many fields due to its excellent properties such as slender, strong, white, and shiny. However, the fiber is wrapped by the gum materials with hemicellulose and pectin as the main components, and degumming is the pivotal process for obtaining the refined ramie fibers. Using a single strain for ramie degumming, the gum removal rate is always low due to its incomplete degumming enzyme system. As a result, lots of alkalis and bleachers have to be used to remove the residual gum materials in the later stage. [Objective] Enriching the key enzyme systems in the degumming process of ramie fibers, improving the gum removal rate, reducing the usage of chemical reagents in the later stage, and promoting the industrial application of ramie biological degumming. [Methods] One strain Bacillus sp. HG-9 with highly activities of pectinase and mannanase and another Bacillus sp. HG-25 with highly activities of xylanase were selected to establish a mixed microbial degumming technology. Besides, the related parameters of the degumming process were optimized.[Results] When the inoculation amount of the two strains, the ratio of water to material, the initial pH value, the temperature, and the degumming time were 6%, 16:1, 5.9, 37.6℃, and 14 h, respectively, the effect was best. Compared with strain HG-9 alone, the degumming time was reduced by 2 h, and the removal rate of gums, hemicellulose, and lignin were increased by 9.32%, 21.24%, and 17.93%, respectively. The amount of sodium hypochlorite was reduced by 20%. The results of scanning electron microscopy showed that the fibers obtained by composite degumming was smoother, without obvious distortion and damage. Meanwhile, the fiber dispersion was high. [Conclusion] With the synergistic action of composite microorganisms, the key enzymes in the degumming process was enriched, the gum removal rate was improved, the degumming time and the amount of bleacher in the later stage was reduced. This study provided meaningful guidance for the further industrial application of ramie biological degumming.

Abstract:[Background] β-glucosidase (EC3.2.1.21) is one of the important components of the three cellulase enzymes. At present, most of the industrial cellulase are derived from fungi such as Trichoderma, and less derived from bacteria, and there are still problems in application such as narrow application range of reaction conditions (such as temperature, pH), low enzyme activity, and high acquisition cost. These greatly limit the application of β-glucosidase. Screening β-glucosidase from soil bacteria has a great possibility to screen out enzymes with better enzymatic properties, thus solving existing industrial problems. [Objective] Use functional screening method to screen β-glucosidase from soil, obtain a new type of β-glucosidase through gene recombination, expression optimization and protein purification, explore its enzymatic properties, and its industrial application lay the foundation. [Methods] The β-glucosidase was screened from the soil by the functional screening method. Because its full length is 747 bp, it was named Bgl747, and the recombinant expression plasmid pET-28a-Bgl747 was constructed with Escherichia coli BL21(DE3). Induced by IPTG to achieve soluble expression and optimize expression conditions, the purified enzyme was purified by His-tag protein purification kit, and its enzymatic properties were explored. [Results] β-glucosidase Bgl747 as part of the BglB superfamily, its molecular weight is 27.23 kD, and an optimal pH of 4.0, an optimal reaction temperature of 45℃; The optimal induction conditions are:when OD₆₀₀ 1.0, add the final concentration IPTG 0.6 mmol/L, the highest expression level of β-glucosidase Bgl747 protein was 1.82 mg/mL after being induced at 37℃ and 220 r/min for 10 h. The specific enzyme activity when the substrate is p-nitrophenyl-β-D- galactopyranoside (pNPG) is 225.07 U/mg, the Michaelis constant K_m value and the maximum reaction rate are respectively 0.268 mmol/L, 547.23 μmol/(L·min); 1 mmol/L K⁺, 1 mmol/L and 10 mmol/L Fe²⁺, 30% methanol, 30% ethanol, 1 mmol/L and 10 mmol/L guanidine hydrochloride all have a promoting effect on enzyme activity, 30% TritonX-100 and 10 mmol/L SDS inhibits enzyme activity more obviously; The enzyme is feedback inhibition by the product glucose, the greater the glucose concentration, the more obvious the inhibitory effect, but when the glucose concentration is 1 mol/L, the enzyme activity remains above 50%. [Conclusion] Bgl747 has a wide and stable reaction temperature range and excellent enzymatic properties, and lays the foundation for its industrial applications such as cellulose degradation.

Abstract:[Background] Agaro-oligosaccharides (AOS) have become a research hotspot in cosmetics, food, medicine and other fields. Biological enzyme method is considered as an efficient method to prepare AOS. [Objective] The agarase gene aga2660 was obtained from Flammeovirga pacifica WPAGA1. The gene aga2660 was cloned and transformed to be expressed in Escherichia coli. The properties of recombinant enzyme and enzymatic hydrolysate were analyzed. [Methods] The pure expression product was obtained by clone expression and nickel column purification. Enzymatic products of the agarase were analyzed by thin-layer chromatography (TLC) and ion chromatography (IC). The optimization of enzyme production conditions was carried out in a 5 L fermentor using the strategies of exponential feeding in the feeding stage and continuous feeding of lactose in the induction stage. [Results] The elected gene aga2660 possessed typical sequence characteristics of glucoside hydrolase family 50 (GH50). The end-product of agar degradation by Aga2660 was neoagarobiose. The optimum temperature and pH of Aga2660 was 30℃ and 7.0, respectively. Meanwhile, Aga2660 showed outstanding temperature and pH stability. Activities of Aga2660 were enhanced by Mn²⁺, Ca²⁺ and Mg²⁺ (1 mmol/L). The enzyme activity reached 11.81 U/mL after fermentation optimization strategy, which was 13.2 times higher than that before optimization. [Conclusion] The agarase Aga2660 was an agarase of GH50 family and indentified with high agar-degrading activity and excellent stability against acid, alkali and thermo. Its enzymolysis product is neoagarobiose, which lays a good foundation for large-scale preparation of AOS with single polymerization degree.

Abstract:[Background] The tomb murals preserved at the original sites are generally threatened by the microbial disease, and the long-term controlling of these microorganisms is a long-standing problem in the field of cultural heritage conservation. [Objective] we aimed to explore the cultivable fungal diversity on moldy murals of the tomb corridor surfaces, to isolate the dominant fungal strains and screen the long-acting biocides, and provide a scientific support for the control of the mural's fungal disease of this tomb. [Methods] Mural samples with whitish moldy necrosis were carefully collected by sterile swabs. The surface morphology of samples was analyzed by scanning electron microscope (SEM). The culture-dependent method was employed to isolate fungal strains, and combine with molecular techniques for identification, the community composition of fungi was analyzed thereafter. Combined with the laboratory inhibition zone test and the in-situ biocides test, the size of the inhibition zone of different biocides, the in-situ cultivable microbial concentration after biocides application, and the ATP fluorescence values differences were analyzed in order to find a lasting efficacy of the biocides. [Results] There were a large amount of mycelia with conidia, the culturable fungi in the white mycelium samples affilliated to the six genera of Ascomycota phylum, the dominant cultivated fungus was Parengyodontium album (98.13%). After screening and evaluation in laboratory and in-situ tests, it is clear that the most effective biocide was dichlorophene compounds (0.5% dichlorophene with 75% ethanol), and no repeated outbreaks of microorganisms occurred during the 7-year consecutive monitoring period. [Conclusion] P. album was the dominant cultivable fungi that caused mural moldy at tomb corridor. Dichlorophene compound biocide used in the controlling test of Xu Xianxiu's tomb mural of Northern Qi dynasty has achieved the longest timeliness of maintenance; and it is recommended to combine the emergency protection, environmental regulation and follow-up monitoring in the future, as a result, to achieve long-term prevention and control of the microbial hazards to the tomb murals.

Abstract:[Background] With the rapid development of the lactic acid bacteria (LAB) industry, it is becoming a research hotspot in the fermented field to analyse the community structure of LAB in different habitats and to develop and utilize bacterial resources. It is of great significance to isolate and preserve of LAB for the screening and utilization of excellent bacterial species. [Objective] In order to clarify the composition of LAB in the birch bark of the primitive forests of Inner Mongolia, and to obtain wild LAB strains, and to provide core strains for excellent strains screening and industrial production of LAB, we isolated and identified and screened LAB from bark of birch trees in Primeval Forest and also survey their characteristics. [Methods] Traditional pure culture method was used to isolate and purify the endogenous and epiphytic LAB from 20 samples of birch bark. The species identification were performed by 16S rRNA gene sequence analysis and the phylogenetic relationship study method. The dominant strains were screened, and 2 strains with excellent characteristics were obtained. [Results] 16S rRNA gene sequence analysis showed that the 112 isolates of LAB were identified as 3 genera and 7 species, of which 53 epiphytic isolates and 59 endophytic isolates, including Enterococcus mundtii (27 strains), Enterococcus faecium (16 strains), Enterococcus faecalis (12 strains), Enterococcus durans (1 strain), Lactococcus lactis (37 strains), Lactococcus garvieae (18 strains), Lactobacillus fermentum (1 strain). Among 37 strains of Lactococcus lactis, 2 strains IMAU98457 and IMAU98428 with higher acid production and lower pH were obtained after preliminary and re-screening by comparing the pH and lactic acid production of different LAB under the same conditions. The biological characteristics of the strains were studied and it was found that the optimum growth temperature of strains IMAU98457 and IMAU98428 was 37℃, and the suitable pH range was 5.5−7.5. After the fermentation at 37℃ for 18 hours, the OD₆₀₀ value of the bacterial solution was as high as 2.531 0 and 2.518 2 respectively. The pH was as low as 4.36 and 4.34 (total acid) respectively, and the strains IMAU98457 and IMAU98428 have certain salt tolerance. When the NaCl concentration is greater than 6%, the growth of the strain is significantly inhibited. [Conclusion] Among the 112 lactic acid bacteria isolated from 20 birch bark samples, Lactococcus lactis was the dominant strain of birch bark samples in Chifeng, Inner Mongolia, with a total of 37 strains, accounting for 33% of the total isolates. Compared with endophytic samples, endophytic samples had more abundant species resources than epigenetic samples, and there were great differences in dominant bacteria between endophytic samples and endophytic samples. Two strains IMAU98457 and IMAU98428 with fast propagation rate, high acid production rate and low pH were screened from the dominant strain Lactococcus lactis.

Abstract:[Background] Cyanophages, are viruses infecting cyanobacteria and a potential factor to control the growth and elimination of cyanobacteria blooms, which are of great significance to the regulation of cyanobacteria community structure. A large number of studies previously reported have revealed the high diversity of cyanophages in marine and freshwater environments, however, the knowledge of cyanophages in wetlands is scant. [Objective] It is necessary to clarify the genetic diversity of cyanophages gene g20 in Napahai plateau wetland in China, and provide a theoretical basis for further research on microbial resources and ecological functions of plateau wetlands. [Methods] The water samples were collected during the rainy season. The capsid protein gene g20 as marker gene was amplified by PCR with specific primers Cps1/Cps8 in this study, and 26 different effective sequences of gene g20 were obtained. The principal coordinates analysis and phylogenetic analysis for gene g20 sequences from above and other ecological environment were performed. [Results] The phytophagy sequences in Napahai plateau wetland were more closely related to rice field sequences compared with other marine and freshwater phytophagy sequences. However, some of the sequences were clustered separately, they may be unique type of cyanophages in Napahai plateau wetland. [Conclusion] It shows that cyanophages is abundant and show unique in Napahai plateau wetland.

Abstract:[Background] As high concentration organic wastewater, piggery wastewater was one of the main factors that lead to area-source pollution of agriculture in China. At present, an increasing number of researchers focused on piggery wastewater treatment with bacteria-algae symbiotic system. Compared with the traditional sequencing batch reactor (SBR), the algal-assisted SBR showed better performance in nitrogen and phosphorus removal, sludge activity improvement and energy consumption reduction. [Objective] Aiming at the influence of algal-bacterial symbiosis system on the nitrogen and phosphorus removal, the distinction on sludge characteristics and microbial community structure were anlyzed between the algal-bacterial symbiosis system and the conventional SBR. [Methods] The algal-assisted SBR (R1) and the conventional SBR (R2) were operated in parallel at room temperature to study the performance of piggery wastewater treatment. The sludge characteristics were also observed, such as sludge particle size, sludge settleability and microbial metabolites. Denaturing gradient gel electrophoresis (DGGE) was used to analyze the microbial community structure in R1 and R2 systems. [Results] compared to the R2, chemical oxygen demand (COD), NH₄⁺-N, total nitrogen (TN) and total phosphorus (TP) were increased by 5.1%, 20.3%, 19.4% and 23.9%. The average values of extracellular polymeric substances (EPS) and soluble microbial product (SMP) in the R1 were 3.7% and 38.5% higher than that in the R2, respectively. Compared to that of R2, the sludge particle size of R1 was increased by14.8%, due to the adhesion of algal to the sludge. In addition, the SVI value of R1 was 11.7% less than that of R2, indicating a poor settleability of sludge in the R1. The sludge specific oxygen uptake rate (SOUR) of R1 was 64.8% higher than that of R2. The formation of stable bacteria algae symbiosis system further reduced the concentration of suspended solids in the effluent of the R1, which further indicated that the addition of algae could improve the characteristics of R1 sludge. [Conclusion] Actinobacteria, Alphaproteobacteria and Gammaproteobacteria as the dominant species in the R1 played an important role in the piggery wastewater treatment. The major Chlorophyta were Desmodesmus and Acutodesmus which had a significant effect on nitrogen and phosphorus removal.

Abstract:[Background] Combined plant-microbial remediation technology plays an important role in reducing environmental stress, enhancing plant resistance and improving mining landscape. Rhizosphere microorganisms can interact with plants which can promote plant growth and enhance the tolerance of plant on abiotic stress. [Objective] To identify two strains of rhizosphere bacteria isolated from copper tailings, in order to study the growth-promoting properties of the two strains and determine the influence of the two strains on V. zizanioides grown on coal gangue. [Methods] 16S rRNA gene sequence identification and scanning electron microscope observation were performed on P5-11 and P5-19, the growth-promoting properties of the strains were measured. The strains were inoculated into V. zizanioides grown in coal gangue covered with 5 cm soil and coal gangue mixed with 10% soil. After two months, the physical and chemical indexes and physiological indexes of V. zizanioides were measured. [Results] P5-11 and P5-19 were Herbaspirillum, which had the functions of nitrogen fixation, phosphate-dissolving, produced indole-3-acetic acid (IAA) and siderophore. Among them, the IAA production capacity of P5-19 was about twice that of P5-11, and it had better growth-promoting properties. Herbaspirillum inoculation could improve plant height, biomass, total nitrogen accumulation and the antioxidant enzyme activity, and reduce the malondialdehyde accumulation. [Conclusion] The two strains of Herbaspirillum had good growth-promoting properties, which could promote the growth of V. zizanioides under coal gangue stress, which not only provided excellent strains for the preparation of microbial fertilizer, but also provided reference value for the application of V. zizanioides in the ecological restoration of mining areas.

Abstract:[Background] At present, the characteristics of Δ6 fatty acid desaturase (FADS6) from various species have been identified through the yeast expression system. Since FADS6 is a multiple transmembrane protein, it is challenging to achieve large-scale expression and purification. [Objective] To construct a high-efficiency expression strategy of FADS6, the present study will analyze the influence of the location of the purification tag on heterologous expression of Mortierella alpina FADS6I (MaFADS6I). [Methods] Tandem affinity tag HRV 3C-Protein A-His was added into the Pichia pastoris vector, followed by the insertion of MaFADS6I sequence to construct recombinant vectors with the N-terminal or C-terminal tag, respectively. Recombinants were obtained through electro-transformation. The protein expression level of MaFADS6I in recombinant strains was analyzed by dot blot hybridization (dot blot), polyacrylamide gel electrophoresis (SDS-PAGE) and western blot, and the fatty acids catalyzed by MaFADS6I was detected by gas chromatography-mass spectrometry (GC-MS). [Results] Transformants with different MaFADS6I expression levels and catalytic activities were obtained. compared with the N-terminal tag, the C-terminal tag was more conducive for the expression and catalytic activity of MaFADS6I. [Conclusion] MaFADS6I with C-terminal purification tag is more conducive to the expression of the protein in the yeast system and the conversion of substrates than with N-terminal tag, providing a foundation for the high-efficiency expression and structural and functional studies of FADS6.

Abstract:[Background] As a substitute of Cordyceps sinensis, Cordyceps militaris has similar pharmacological activities with Cordyceps sinensis. It is rich in protein and amino acids, which is usually used as an important index to measure the nutritional value of fungi. It has become a research focus to isolate and purify proteins or peptides with potential clinical application value from Cordyceps militaris. [Objective] The protein composition of fruiting bodies of wild and commercial strains of Cordyceps militaris in Shenyang were detected, and the differences of protein types, quantities and functions under the same cultivation conditions were analyzed, which provided the proteomic data basis for further study and identification of protein for medicinal use and targeted domestication of northern caterpillar in Shenyang area. [Methods] Wild Cordyceps militaris strains were collected from Qipan Mountain of Shenyang. Fruiting bodies of wild and artificially cultivated Cordyceps militaris strains were obtained by tissue isolation and liquid fermentation respectively at the same time. The quantitative proteome of fruit body samples from wild and commercial sources was studied by non-standard quantitative technology liquid chromatography-mass spectrometry after protein extraction and trypsin enzymolysis. [Results] A total of 9 233 specific peptide fragments and 1 923 proteins were identified, including 1 163 quantifiable proteins. 214 proteins were up-regulated and 181 proteins were down-regulated in fruit bodies cultured from wild sources. After functional enrichment analysis, these differential proteins were mainly involved in energy production/conversion, amino acid transport/metabolism and antioxidant function. Under the same nutritional conditions, the expression of protein related to energy metabolism and amino acid metabolism of wild-type strains was higher than that of commercial strains, and the expression of an important antioxidant protein (Gene Name:ISF_02112) from wild cultivated strains was much higher than that in commercial strains (fold change>9). There were 22 differentially expressed proteins related to both antioxidant and metabolic functions. [Conclusion] Some excellent biological characteristics of wild Cordyceps militaris in Shenyang can be preserved by proper artificial cultivation. The fruiting bodies cultivated by the two strains have rich and excellent antioxidant proteins, and the antioxidant capacity of fruiting body proteins is related to their overall metabolic capacity. The results of this study provide the basis of proteomics data for further study and identification of the herbal protein and targeted domestication of Cordyceps militaris.

Abstract:[Background] Many domestic studies have reported in detail the application of gene knockout with suicide vectors in single gene knockout. However, the changes in bacterial resistance after gene knockout and the impact on subsequent gene knockout remain unclear. [Objective] In order to explore the changes in the resistance of Vibrio vulnificus (V. vulnificus) to chloramphenicol and its effect on subsequent gene knockout in the process of gene knockout with pDS132, the vvhA and rtxA1 gene knockout strains were constructed. [Methods] The single gene knockout strains YJ016-ΔvvhA, YJ016-ΔrtxA1 and double gene knockout strains YJ016-ΔvvhAΔrtxA1 of V. vulnificus YJ016 were constructed by the homologous recombination with suicide vector pDS132. The concentrations of chloramphenicol and the proportions of strains successfully recombined on the screening plate were recorded. The minimum inhibitory concentration (MIC) of each strain to chloramphenicol and other antibiotics, and the mutation frequency of chloramphenicol resistance were determined and then analyzed.[Results] The MIC and the mutation frequency of chloramphenicol of the single gene knockout strains were higher than that of the wild type; the diameter of the gentamicin inhibition zone of the double gene knockout strains was smaller than that of the wild type. When the rtxA1 gene was knocked out on YJ016-ΔvvhA and the chloramphenicol concentration of was 2, 4 μg/mL, the proportions of strains with single-crossover recombination were 40% (8/20) and 5% (1/20), respectively; when the vvhA gene was knocked out on YJ016-ΔrtxA1, the proportion was 0% (0/20). [Conclusion] In the process of gene knockout with the suicide vector pDS132, the resistance of V. vulnificus to chloramphenicol increased, which may affect the subsequent screening in single-crossover recombination. The result is helpful for the application of the homologous recombination technique with suicide vector to multiple gene knockout.

Abstract:[Background] Tobacco wild fire is one of the main diseases on tobacco. It is a promising method to control tobacco wild fire with antagonistic bacteria. [Objective] To analyze the composition and diversity of microbial community in tobacco leaf, and explain the control effect of antagonistic bacteria on tobacco wild fire. [Methods] Three antagonistic groups were applied to tobacco, and the effects of antagonistic groups on the structure and diversity of tobacco leaf microbial community were analyzed by 16S rRNA gene high-throughput sequencing and bioinformatics.[Results] The control effect of antagonistic bacteria on tobacco wild fire reached 50.44%−68.58%. The community structure and composition of antagonistic groups had significant changes, was significantly higher community diversity, compared with the control group. After treatment with antagonistic flora, the proportion of tobacco leaf microflora, such as Pantoea, Stenotrophomonas and Pseudomonas, changed significantly, Bacillus and Stenotrophomonas increased by 3.9 and 7.02 times, respectively, compared with the control. The abundance was negatively correlated with disease index. [Conclusion] The antagonistic bacteria had a good control effect on tobacco wild fire. The composition and diversity of the microbial community in the tobacco leaves were significantly affected by the application of antagonistic bacteria. The dominant bacteria such as Pseudomonas and Stenotrophomonas could colonize in the tobacco leaves and play a role in controlling tobacco wild fire.

Abstract:[Background] In recent years, the degree of soil salinization in Daqing has been gradually intensified. microbial improvement of saline-alkali soil is a hot research topic now. [Objective] Saline-alkali tolerance bacteria were screened out from Daqing saline-alkali soil, verify its growth-promoting effect, providing microbial resources for improving the saline-alkali soil in Daqing. [Methods] Screening mediums were used to separate saline-alkali tolerance and growth-promoting bacteria from Daqing saline-alkali, the strains were identified by morphological observation, physiological and biochemical identification and 16S rRNA gene sequence analysis, the effect of strains on mung bean growth and soil bacterial community structure under saline-alkali stress was tested. [Results] A saline-alkali tolerance and growth-promoting strain DQSA1 was screened, which could fix nitrogen and produce ACC deaminase, siderophore and indole-3-acetic acid (IAA), and identified through physiological and biochemical identification and phylogenetic analysis as Zobellella. Inoculation of strain DQSA1 after planting mung beans in saline soil, the fresh root weight, root dry weight and chlorophyll content of mung bean after treatment increased significantly, increased by 33%, 32% and 79% respectively. strain DQSA1 could significantly increase the content of soluble sugar, proline and soluble protein in mung bean leaves, increase by 10%, 80% and 73%, respectively. Compared with the content of CK, the proline content and soluble protein content of mung bean roots were increased by 78% and 44%, respectively. High-throughput sequencing was performed on the soil in which mung bean were grown, the results showed that the strain DQSA1 can colonization in a saline-alkali environment and promote the growth of beneficial Rhizobium spp. and Sphingosine spp. [Conclusion] strain DQSA1 could change the soil bacterial and promote plant growth under saline-alkali conditions, providing effective microbial resources for improving saline-alkali soil.

Abstract:[Background] Paris polyphylla var. chinensis is a kind of precious chinese medicine materials. Over-exploitation leads to the lack of wild resources. The artificial cultivation is restricted by the poor growth and frequent occurrence of diseases. [Objective] Exploring of plant probiotics is an effective and environment friendly solution, according to the requirements of ecological planting. [Methods] Endophytic fungus was isolated by conventional methods and identified by comprehensive methods. The germination test of maize seeds was carried out for screening the isolated fungus which had been reported to promote growth and resist disease. The nitrogen fixation activity of microorganism was tested qualitatively by nitrogen-free ager. Antimicrobial activity was detected by plate antagonism test. High performance liquid chromatography-mass spectrometry (LC-MS) were used to detected the metabolic components of microorganism and the contents of salicylic acid, jasmonic acid, abscisic acid, gibberellins, cytokinins, auxins in leaves. The underground biomass indexes were also measured. [Results] One of fungi was identified as Aspergillus sydowii by rDNA ITS sequencing, morphological observation, physiological and biochemistry identification, named jdqmzz-1. The strain jdqmzz-1 which contains kinds of stimulative, antibacterial, and insecticidal substances can promote the growth of Paris polyphylla var. chinensis, fix nitrogen, and antagonize pathogenic microorganism. The antagonistic index was 42.50%. The Paris polyphylla var. chinensis seedlings endogenous gibberellins and auxins in leaves were increased by 54.1 and 2.3 times, chlorophyll contents were increased by 48.80%, malondialdehyde contents were decreased by 15.20%, respectively, while treating with the strain. Average root numbers, root lengths and hundred plant weights of seedlings were also significantly increased. [Conclusion] The isolated endophytic fungus Aspergillus sydowii strain jdqmzz-1 can effectively promote the growth of Paris polyphylla var. chinensis.

Abstract:[Background] Straw biodegradation has been the focus of increasing amounts of attention because of its high degradation efficiency and environmental friendliness. [Objective] The key functional microbes in the process of straw degradation of composite microbial system GF-20 and their relationship with the characteristics of degradation were clarified. [Methods] GF-20 was cultured at 10℃, and the dynamics of growth and straw decomposition characteristics were determined, enabling the estimation of corn stalk decomposition by GF-20. Furthermore, the microbial community succession in different degradation periods was analyzed using MiSeq high-throughput sequencing technique. [Results] The composite microbial system GF-20 grew logarithmically, the pH value rapidly dropped to 6.98, and the total sugar content of the fermentation system quickly decreased to 0.22 mg/mL within 1 d after inoculation. The soluble chemical oxygen demand declined to 4.77 g/L after 3 d, and the oxidation-reduction potential rapidly reduced to -303 mV. The GF-20 efficiently secreted cellulose, and rate of degradation of corn stalk, lignin, cellulose and hemicellulose were 31.97%, 32.30%, 44.85% and 43.84%, respectively, after 15 days of fermentation at 10℃. The dominant genera included Cellvibrio (29.55%), Chryseobacterium (7.35%), Hydrogenophaga (3.86%) and Pseudomonas (3.42%) during the initial stage (2 d, 5 d) and the key functional microbes Azospirillum (9.92%), Rhizobium (6.99%), Nubsella (5.06%) and Stenotrophomonas (3.37%) during the mid-term stage (7 d, 10 d). The abundances of Taibaiella (13.82%), Pleomorphomonas (13.69%), Flavobacterium (14.89%), Cellulomonas (7.18%), Devosia (7.36%), Pedobacter (4.32%) and Sphingomonas (2.23%) increased significantly during the late stage (12 d, 15 d). [Conclusion] The dynamic changes in bacterial consortium structure and the characteristics of straw degradation during different periods of degradation were clarified and provide a theoretical basis for the rational utilization of composite microbial system GF-20.

Abstract:[Background] Mycoplasma hyopneumoniae is an important pathogen in pigs. The research tools of this bacterium are few, especially the antibody that is used to carry out the research on its pathogenicity. [Objective] To prepare the polyclonal antibody against Mhp366-N protein of M. hyopneumoniae and determine its application and the optimal dilution. [Methods] Expression of Mhp366-N protein was induced by recombinant bacterium E. coli BL21(DE3)-pET28a(+)-mhp366-N, and the protein was purified. The purified protein was used to immunize mice to prepare polyclonal antibody. Using Mhp366-N polyclonal antibody as the primary antibody, the Mhp366 protein in 3D4/21 cells infected with M. hyopneumoniae AH strain was detected by Western blot and immunofluorescence, and the optimal dilution of antibody in each assay was determined. Then, M. hyopneumoniae was detected in alveolar macrophages collected clinically. Finally, the presence of M. hyopneumoniae in lung cells was detected by immunohistochemistry. [Results] The purity of purified Mhp366-N protein was more than 85%, and the titer of Mhp366-N antiserum was between 1:128 000 and 1:512 000. The optimal dilution of Mhp366-N polyclonal antibody in Western blot was 1:100 000, and in immunofluorescence assay was 1:1 000−1:10 000 000. In addition, the polyclonal antibody could be used to detect M. hyopneumoniae in porcine alveolar macrophages and cell lines. The results of immunohistochemistry showed that M. hyopneumoniae could enter porcine alveolar macrophages, type I and II alveolar epithelial cells. [Conclusion] Preparation of Mhp366-N polyclonal antibody could provide a good research tool for the research on the pathogenesis of M. hyopneumoniae.

Abstract:[Background] The infection rate of Mycoplasma synoviae (MS) in chicken flocks has been increasing in China since 2010, and MS has been widely present in different flocks, including laying hens and/or breeders, white feather broilers and local breeds. The positive rate of MS antibody has exceeded 40%, which has seriously harmed Chinese chicken industry and caused serious economic losses. [Objective] The pathogenicity of MS to 56-day-old specific pathogen free (SPF) chickens with different infection routes was simultaneously compared. [Methods] The MS virulent FZ strain was used to infect 56-day-old SPF chickens by eye dropping, footpad injection, chest subcutaneous injection, single tracheal injection, and three consecutive tracheal injections. The clinical symptoms and anatomical pathological changes were observed from infection chickens, and MS antibody, re-isolation of pathogens from the trachea, and histopathological was determined at post-infection. [Results] The chicken infected MS FZ strain with different routes had different clinical symptoms and different morbidity. Footpad injection and chest subcutaneous injection could cause 100% of chickens exhibiting footpad swelling or chest cyst, while a single or three consecutive tracheal injection could cause 33%−50% of chickens exhibiting severe air sacculitis, the eye dropping infection had hardly caused clinical pathological changes; The footpad swelling showed proliferation of granulation tissue and a large amount of yellow cheese-like mass, and some blood-red liquid and yellow cheese-like mass were found in the chest cyst; Histopathological results showed that three consecutive tracheal injection were more likely to cause damage to the trachea, manifested mild inflammatory cell infiltration in the lamina propria/submucosa of the trachea, footpad swelling and chest cyst tissue had a lot of fibers tissue and blood vessel hyperplasia, accompanied by a large number of inflammatory cell infiltration. It was found that MS re-isolation from tracheal of birds challenged by eye dropping and tracheal injection could reach to 100%, and the MS were also isolated from the trachea by chest subcutaneous or footpad injection. MS antibody was easier to be detected from chickens challenged by footpads. [Conclusion] The pathogenicity of MS to SPF chickens with different infection routes was systematically compared, then the typical pathological lesions or virulence evaluation methods were presented after MS artificial infection, and a model of MS artificial infection to 56-day-old SPF chickens was successfully established. It is found that MS re-isolation from trachea was main indication for eye dropping and tracheal injection, and air sacculitis was auxiliary. In addition, footpad swelling or chest cyst was cardinal symptom to footpad and chest subcutaneous injection, respectively.

Abstract:[Background] Salmonella is a common zoonotic pathogen. With the increasing number of pet owners, the threat of Salmonella in pet intestines to public health is gradually emerging, but there are few reports on the epidemiology and antimicrobial resistance of pet-associated Salmonella.[Objective] To investigate the epidemiology, antimicrobial susceptibility to commonly used antimicrobials and the prevalence of extended-spectrum β-lactamases (ESBL) gene and plasmid-mediated quinolone resistance (PMQR) genes of Salmonella isolated from pets in Beibei District, Chongqing. [Methods] Salmonella was isolated and identified by pre-enrichment, selective enrichment, selective plate screening and amplifying invA gene through PCR. Then, the antimicrobial susceptibility of the isolates to 28 antimicrobials was determined, and 10 ESBL genes and 10 PMQR genes were detected. [Results] A total of 41 Salmonella strains were isolated, the isolation rate was 3.95%. The resistance rates of these strains to sulfamethoxazole, ampicillin, tetracycline, doxycycline were more than 50%, 82.92% of them were multi-drug resistant, and all of isolates were completely sensitive to amikacin, ofloxacin, enoxacin and gatifloxacin. 85.37% of the isolates carried ESBL gene and the bla_TEM gene was the most popular. 46.34% of the isolates had PMQR genes and qnrS was the most popular one. 48.57% of ESBL positive strains carried at least one PMQR gene. [Conclusion] The above results demonstrated that pet-associated Salmonella in Beibei District were resistant to a variety of antimicrobials, and the drug resistance may be mediated by drug-resistant plasmids.

Abstract:[Background] At present, there are few studies on how to solve the lethal pathogenicity of harmful fungi to Drosophila melanogaster. The intestinal symbiotic bacteria of Drosophila are one of the current research hotspots, and the study on the interaction between symbiotic bacteria and harmful fungi has attracted widespread attention. [Objective] To assess the competitive interaction between Drosophila symbionts and pathogenic fungi, consequently mitigating the toxicity of fungi to their hosts. [Methods] Fungi were isolated from fly food using PDA medium. The interaction between Drosophila symbionts and pathogenic fungi was examined by means of colony diameter, the number of spores and mycelium branches. The pathogenicity of fungi was examined using Drosophila infection. The germ-free and gnotobiotic flies were established to verify the protection of Drosophila symbionts against fungi. The 2-choice egg-laying apparatus was employed to assay the oviposition selection of D. melanogaster female adults. [Results] A fungus was identified as Phomopsis FY that was detrimental to the survival and development of Drosophila upon infection. Acetobacter orientalis hindered the growth of Phomopsis FY in vitro, and decreased the mortality rate of Phomopsis FY-infected flies in vivo, consequently mitigating the toxicity of Phomopsis FY to the hosts. Additionally, the presence of A. orientalis overrode the avoidance of oviposition on Phomopsis FY-associated substrates. [Conclusion] Phomopsis FY was identified as a conditionally potential Drosophila pathogen. Commensal A. orientalis mitigated the susceptibility of Drosophila to pathogenic fungi, providing insight into the natural interplay between commensal and pathogenic microbial communities that contribute to animal health and pathogenesis.

Abstract:[Background] Nocardia seriolae is a kind of Gram-positive aerobic bacterium, which may cause sarcoidosis in both freshwater and marine fish, among them, perciformes are the most susceptible to the disease. In recent years, serious losses caused by N. seriolae in freshwater fishes such as Micropterus salmoides and Channa argus have been increasing. [Objective] To compare and analyze the homology, growth characteristics, pathogenicity and antimicrobial susceptibilities, 9 N. seriolae strains isolated from different freshwater fish species suffering from sarcoidosis in different years and regions were determined in this study, and to further develop a reliable scientific measures to control and treat this disease. [Methods] 16S rRNA gene and the housekeeping gene secA1 were amplified and sequenced for identification and phylogenetic analysis of N. seriolae after biological characteristics and phylogenetic analysis. PCR-restriction fragment length polymorphism (PCR-RFLP) was developed to analyze the homoology of different origins of N. seriolae. Growth curve of 9 strains was determined by in vitro culture. Pathogenicity of 9 N. seriolae strains was characterized by artificial injection against largemouth bass. Antimicrobial susceptibility testing was carried out to determine the phenotypes of 13 antimiocribal agents by microbroth double dilution method. Virulence genes and drug resistance genes of 9 strains were also detected in this study. [Results] Based on the biological characteristics, most of the phenotypes in 9 strains were similar to Nocardia seriolae. Nine strains were identified to be N. seriolae and clustered into one category with high intraspecies similarity. PCR-RFLP digestion patterns were identical and the growth curvre of 9 strains showed no significant difference of growth characteristics. A variety of virulence genes were detcted in 9 strains. Three genes of mce1A, mig and pup were all detected in 9 strains, whereas dop and whiB3 genes were only carried in some of the strains. There was no significant difference in the pathogenicity of 9 N. seriolae to largemouth bass. All the strains were resistant to sulfamonomethoxine, flumequine and ampicillin, while most of the strains were susceptible to other antimicrobial agents tested. bla_TEM gene was detected in 9 N. seriolae strains, some of which also carried sul1 gene. [Conclusion] Nine strains of N. seriolae isolated from diferent origins showed similar growth characteristics, pathogenicity and antimicrobial susceptibilities. It implied that N. seriolae was the predominant pathogen of sarcoidosis prevailed in freshwater fish and with similar homology. The results of the study provide foundation of further research on the pathogenesis, prevention and control technology of N. seriolae.

Abstract:[Background] Understanding functional microbiota inside crude oil samples is crucial in microbiota design for enhancing oil recovery. [Objective] Identifying potential core functional microbiota from a crude oil sample that have ability to produce acid/gas through metagenomic analysis. [Methods] The compositional and functional changes of the indoor activated crude oil samples were obtained with the metagenomic data. The potential endogenous core functional microbiota related to the production of gas and acid were obtained through bioinformatics, multivariate statistics and network analysis. [Results] The co-abundance analysis deduced that Bacillus licheniformis, along with Coprothermobacter proteolyticus, Marinobacter spp., Anaerbaculum hydrogeniforms and Petrotoga mobilis that clustered as a group, were the core functional bacteria relevant to the change of the pH value. Functional analysis of strains in this group through draft genome assembly indicated that the pathways for acid production, including for lactic acid, acetic acid and formic acid fermentation using pyruvate/acetyl coenzyme A, were enriched. Enterococcus faecium was considered as the core microbe in another group, which had Shinella zoogloeoides, Paracoccus deniticans, Paracoccus spp. and Enterobacter cloacae. Among these, Enterobacter cloacae was positively correlated with total gas production. The assembled bins in this group had the ability to produce nitrogen/sulfur/carbon using nitrate (nitrite)/sulfate (sulfite)/petroleum hydrocarbon. These two groups both had a small number of microbes that have dual ability for gas and acid production and the two groups were negatively correlated. [Conclusion] We had screened out core acid/gas producing microbiota in a crude oil sample through organic activation and metagenomic sequencing technology. These findings provided potential strain targets for further research.

Abstract:[Background] The puffball mushroom is commonly used traditional medicinal edible fungus, which has multiple pharmacological effects, including antimicrobial, anti-inflammatory, anti-tussive, anti-cancer and suppressing cell growth effects. So far, most of the researches on puffball mushrooms have been focused on extraction and pharmacological effect of their bioactive agents. Nevertheless, little is known about the genomic information of puffball mushrooms. [Objective] To acquire genomic information of puffball mushrooms and mine protein-encoding gene of potential application in industry. [Methods] A combination of Illumina and PacBio sequencing technologies was used for genome sequencing, and analysis of genome structure and gene annotation were performed against multiple database, including GO. The sequenced puffball mushroom was subjected to ITS identification, while CAZymes and secondary metabolite biosynthetic gene clusters were identified using dbCAN and antiSMASH, respectively. [Results] The acquired puffball genome was 46.04 Mb in size, encoding 11 903 proteins including GO-annotated genes. Based on the genomic information, the puffball species was identified as Calvatia gigantea using ITS (Internal Transcribed Spacer). A total of 25 genes related to terpene and ergosterol biosynthetic pathway were analyzed, while 317 CAZymes and 18 secondary metabolite biosynthetic gene clusters were identified. In addition, an alpha-amylase from CAZymes was characterized as a potential secretory, tannin-resistant α-amylase. [Conclusion] This study supplied the first sequenced puffball mushroom, and found a potential tannin-resistant α-amylase and multiple new secondary metabolite biosynthetic gene clusters. This study will supply important basic genetic information for genetic evolution and functional researches of puffball mushrooms, their further application in food industry, and development of secondary metabolites of pharmaceutical value.

Abstract:[Background] Tomato fusarium wilt is a common soil born fungal disease in tomato production. [Objective] To identify pectate lyase gene family members in Fusarium oxysporum f. sp. Lycopersici genome and to clarify the expression mode after inoculation.[Methods] PEL family was identified, and the distribution, physical and chemical properties, gene structure, and tertiary structure prediction of PELs in F. oxysporm f. sp. Lycopersici genome were performed by bioinformatics. Expression mode of FoPEL1−16 in inoculated tomato roots were analyzed by RT-qPCR. [Results] There were 16 FoPELs in F. oxysporm f. sp. Lycopersici genome. The amino acid sequences were 163−548 aa in length with 16−21 aa signal peptides. The FoPEL genes were unevenly distributed on 7 chromosomes. 16 FoPELs were divided into four groups according to gene structure and conserved motifs. Evolutionary analysis showed that FoPELs clustered in four clades. Similar domain structure presented in the same family according to tertiary structure prediction. RT-qPCR analysis showed that FoPEL expression levels significantly increased during inoculation. [Conclusion] The pectate lyase existed in the form of gene family in F. oxysporm f. sp. Lycopersici genome, and the difference of gene structure indicated their function diversity. The expression levels of FoPEL were significantly increased during infection, which indicated that FoPEL was involved in the pathogenicity. The study provided a theoretical basis for pathogenic gene function and plant-pathogen interaction of F. oxysporium.

Abstract:[Background] Klebsiella pneumoniae (Kp) is a major pathogen that causes nosocomial infections and community-acquired infections. In recent years, drug resistance in Kp has become more and more serious, making its treatment less effective. So it becomes a challenge in clinical treatment. At present, more unusual cases of pyomyositis caused by Kp have been reported. Infection in deep muscle may form an anaerobic environment. The molybdate transporter ModABC is essential for bacterial anaerobic nitrate respiration. Previous studies showed that ModA might be positively correlated with Kp virulence. [Objective] To investigate the effect of molybdate transporter ModABC on Kp muscle infection and the potential use of tungstate in treatment. [Methods] The modA traceless deletion mutant and complementary strain were constructed. Then the effect and mechanism of ModABC on Kp muscle infection were analyzed by monitoring anaerobic growth, nitrate reductase activity in vitro and mouse muscle infection experiments in vivo. The possibility and efficacy of tungstate treatment for Kp muscle infection were discussed in this study. [Results] The modA traceless deletion mutant and complementary strain were successfully constructed. This study found that deletion of modA led to significantly inhibited anaerobic growth of Kp and reduced nitrate reductase activity in vitro. The knockout strain ΔmodA showed greatly reduced muscle abscess, intramuscular growth and invasion in mouse muscle abscess model. In vitro tungstate treatment inhibited the anaerobic nitrate respiration of wild strain WT, and also significantly inhibited the growth of WT in muscle abscess model, but had no influence on strain ΔmodA. [Conclusion] The molybdate transporter ModABC contributes to Kp muscle infection by providing fitness advantage through enhancing invasiveness and promoting anaerobic nitrate respiration. Tungstate can reduce the fitness advantage conferred by ModA and has a certain therapeutic effect on Kp muscle infection. This study is helpful to elucidate the mechanism of molybdenum's effect on the pathogenicity of Kp. It also provides new insight into the treatment, laying a foundation for further studies, especially carbapenem-resistant hypervirulent Kp infection.

Abstract:Microorganism is ubiquitous in nature, especially foodborne pathogens that cause foodborne diseases. Traditional methods have been already difficult to fit the demands of disease prevention and supervision in the routine work of regulatory authority, so rapid and sensitive high-throughput detection technique has become a research hot spot. Based on the rapid and accurate recognition of microorganism, bi-functional antibodies, aptamers- and phage-based assays are introduced. In terms of multi-target detection, there are three methods that include multiplex PCR, matrix-assisted laser desorption ionization-time of flight mass spectrometry, and biochip. This paper systematically discussed the detection mechanism, advantages and disadvantages, and research progress of each method, hoping to provide theoretical guarantees and reference for the development of rapid high-throughput method for detecting microorganism in the future.

Abstract:Bacillus subtilis is an important model strain widely used in basic research and industrial production, which has many advantages such as non-pathogenicity, the strong capability of secreting proteins, clear genetic background, so it is an ideal host for expressing and secreting heterologous proteins. At present, there are many heterologous proteins that have been expressed and secreted in Bacillus subtilis, which includes valuable proteins such as amylase, beta-mannanase and protease. Based on the key steps of the expression and secretion of heterologous protein, this paper summarizes the traditional strategies and the latest technologies for heterologous protein production in Bacillus subtilis. In addition, we analyze the difficulties in the present research and propose new suggestions and strategies for the improvement of the production of heterologous protein.

Abstract:Honey bees are very important pollinators to agriculture. Gut microbiota is closely related to honey bee health, and affected by many external factors. Herein, the influences on gut microbiota diversity of Apis mellifera ligustica caused by diseases, antibiotics used for disease treatment, and pesticides were analyzed. Furthermore, the researches on application of honey bee gut microbiota as probiotics was summarized. The prospect of research on honey bee and its microbiota is suggested.

Abstract:Natural microorganisms are important factors affecting the physiological growth of grape and the quality of wine, and widely exist in the ecosystem of grape and wine. The species, number and distribution of microorganisms depend on many factors such as climate, soil, growth period and fermentation process control. Under natural conditions, the species composition and metabolism of grape microecosystem directly affect the health of grape vine and the fermentation quality of wine, and produce specific wine terroir. Therefore, the diversity and dynamics of fungal and bacterial communities in vineyard soil, grape berry and wine natural fermentation, as well as the effects of their metabolic enzymes on wine quality were reviewed in this paper, with a view to fully understand the physiological metabolism and ecological function of the microbial community, exploring the interaction mechanism and metabolic function of the grape microecology, and promoting the development of the microbial community in the beneficial direction of grape and wine, in order to achieve the goal of sustainable and high-quality development of grape ecosystem.

Abstract:Organophosphate esters (OPEs) flame retardants/plasticizers have potential adverse effects on human health and are widely detected in various environmental media. To effectively control the pollution of OPEs, green and efficient biodegradation methods have become the research hotspot. The purpose of this paper is to elucidate the biodegradation process and mechanism of OPEs, mainly focusing on the biodegradation pathways and intermediate products of TBP and TPHP. In general, hydrolysis, hydroxylation, and methoxylation are the major biodegradation pathways of OPEs. Cytochrome P450 plays a key role in the process of degradation. Most of the degradation bacteria can mineralize OPEs into inorganic phosphates and other small molecular compounds which are harmless to the environment.

Abstract:Human norovirus is one of foodborne pathogens that causes acute gastroenteritis in human beings around the world. The digestive glands of oysters, mussel and other shellfish have the norovirus receptor analogue, which can accumulate high concentration of virus from the contaminated water. Eating raw or improperly processed shellfish which are contaminated become a major reason for the norovirus infection. The elimination technologies of contaminated shellfish have become a research hotspot in the field of norovirus prevention and control, including the methods of disinfectant, ozone processing, ultraviolet radiation, and probiotics with antiviral effect that recently reported. Infectivity detection of norovirus plays an important role in determining the level of virus contamination in shellfish and evaluating the efficacy of elimination technology. Only complete, infectious virus would threaten human health. In this study, the current researches on detection of norovirus infectivity, product disinfection technology, and elimination technology of shellfish culturing are reviewed, hoping to provide a reference for improving the control technology of foodborne virus.

Abstract:Myxobacteria are a kind of medical microorganisms that could produce abundant secondary metabolites, show complex multicellular behaviors and prey kinds of bacteria and fungi. Therefore, myxobacteria have important values of research and application. However, it seriously impedes myxobacterial mining and utilization because of their difficulties to isolate, cultivate, and research in polyphase classification. This review described myxobacterial features, methods for isolation and purification, and discussed existing problems and improvement measures. In addition, we also analyzed the research status in polyphase classification of myxobacteria. Based on the current technical means and research progress, the future research trends of myxobacteria were prospected. This review aims to provide help to relevant researches on myxobacteria in the future.

Abstract:Bacteria can grow in the environment that other microorganisms can't survive, so it must have more powerful ability to adapt to the external environment. The efficiency of cell signal transduction determines the rate and ability of bacteria to respond to external stimulus. Two-component system is an important structure to maintain the survival of bacteria under conditions of stress. Two-component system Cpx is widely distributed in Gram-negative bacteria, which plays a major role in responding to the changes of external environment and making adaptive response. In this paper, the types of two-component signal transduction system in bacteria, the regulation of Cpx two-component system, the regulated target genes by Cpx two-component system and their physiological behavior were briefly reviewed to provide ideas and theoretical guidance for further research.

Abstract:Nitrate is the main form of nitrogen available to marine microorganisms, and it is also the main limiting factor of nutrients on the production of surface marine organisms. In the ocean, ammonia and nitrite are oxidized to form nitrate. Exploring the niche of nitrite-oxidizing bacteria (NOB) in the marine ecosystem and its response mechanism to marine environmental changes is of great significance to understanding the nitrogen cycle in which microorganisms participate. Here, key bacterial species and their research progress in marine nitrite oxidation are reviewed. The physiological and ecological characteristics of NOB are summarized, and its adaptive strategies in the marine ecosystem are highlighted. Simultaneously, prospective research direction of NOB is elucidated to understand the oxidation process of nitrite in the ocean and the nitrogen cycle in biogeochemistry.

Abstract:As a newly discovered secretion system, type IX secretion system (T9SS) has been found in many species of Gram-negative bacteria. T9SS is involved in the virulence, gliding motility and the degradation of complex biopolymers in bacteria. In recent years, T9SS-related research has always been a hot spot in the field of microbiology. This paper reviews the research on the discovery, components and structures, secretion mechanism and regulation of the T9SS, providing further insight into this highly novel secretion system.

Abstract:The construction and application of online courses has become an important measure in the reform of teaching informatization in colleges and universities at this stage. Effective application of online course resources and the design and practice of blended teaching are the key points in the construction of first-class undergraduate courses. Relying on the online excellent course of microbiology (Shaoxing city, Zhejiang province), the blended teaching based on online course was tried in the science education major of grade 16, 17 and 18. The three rounds of teaching practice showed:the pattern could enhance the students' interest and participation in learning, promote the students' deep learning, and promote the improvement of students' comprehensive ability and quality. The microbiology course has formed a brand at the college and school, and has been widely welcomed and praised by students.

Abstract:[Background] Bluetongue virus (BTV), one kind of arbovirus, causes seriously harms in ruminants and twelve serotypes of BTV (BTV-1, -2, -3, -4, -5, -7, -9, -12, -15, -16, -21 and -24) are widely prevalent in China. [Objective] To provide technical support for the diagnosis and epidemiology research of BTV, we aimed to develop serotyping RT-qPCR method for 12 BTV serotypes which were epidemic in China. [Methods] Primers and TaqMan probes based on the Seg-2 sequences of Chinese BTVs were designed and their specificity and sensitivity were evaluated. The reliability of the established RT-qPCR method was assessed with BTV strains and BTV-positive blood samples then further applied to identify the serotypes of BTV in Culicoides and blood samples. [Results] The established BTV serotype RT-qPCR method showed highly specificity and sensitivity with the amplification efficiency (E) values larger than 90.3%, the correlation coefficient values (R²) ranging from 0.991 to 0.999 and the minimum copy number of detectable BTV nucleic acid ranging from 25 to 48 copies. Serotype identification of 165 isolated BTV strains by RT-qPCR and Seg-2 sequencing showed consistent results. Furthermore, serotype RT-qPCR identification results of 194 BTV-positive blood samples from infected sentinel animals were consistent with serotyping results of the isolated BTVs. Using the established RT-qPCR method, six serotypes of BTV (BTV-1, -2, -4, -5, -16 and -24) were identified from the Culicoides and cattle blood samples collected from Shizong county and Jinghong district of Yunnan province. [Conclusion] The BTV serotype RT-qPCR established here could be used for diagnosis the serotypes of BTV in vectors and animals with advantage of time saving, high specificity and sensitivity.