Abstract:[Background] Vibrio alginolyticus, one of the most important opportunistic pathogens, can cause significant morbidity, mortality and economic losses during shrimp aquaculture. At present, phage therapy acts as an alternative sanitizing agent to control V. alginolyticus. [Objective] To isolate and characterize phage ФV170 lysing V. alginolyticus for the application of the phage to control aquatic animal diseases. [Methods] V. alginolyticus V170 was used as the host strain to isolate phage from Penaeus vannamei aquaculture water. Characterization was done by transmission electro-microscopy, optimal multiplicity of infection, one-step growth curve, lysis spectrum detection, thermal stability, and sensitivity to chloroform. The analyses of phage genome were based on enzyme digestion methods. [Results] A broad-spectrum lytic bacteriophage ФV170 was isolated and the plaques were neat and transparent, with a diameter of 1.5 mm at 12 h. The identification results of transmission electron microscopy (TEM) showed that its head had a stereosymmetric structure of the quasi-diaphedron, with a diameter between 60 nm and 65 nm, and its tail was 65 nm to 75?nm long and 14 nm to 18 nm wide. The nucleic acid type was dsDNA and the genome about 45 kb. Besides, it was not sensitive to chloroform. In conclusion, bacteriophage ФV170 belongs to Caudovirales and Myoviridae. The bacteriophage V170 can lyse 7 of 15?strains of Vibrio alginolyticus, possessing a broad host spectrum within the species. The optimal multiplicity of infection (MOI) was 0.01. One-step growth curve shows that the incubation period of ФV170 was 10?min and the amount of lysis was 101.3. Phage ФV170 was sensitive above 65 °C. [Conclusion] A lytic bacteriophage was isolated with potential functions to treat alginolyticosis in marine aquaculture.

Abstract:[Background] Small non-coding RNAs (sRNAs) play important roles in genetic variation, growth, reproduction, and bacterial pathogenicity, but few studies have been conducted on the opportunistic pathogen of Vibrio alginolyticus. We have identified an sRNA, srvg23535, of V. alginolyticus ZJ-T by RNA-seq and Northern Blot. [Objective] We studied the effect of srvg23535 on the biological characteristics of V. alginolyticus. [Methods] The srvg23535 knock-out mutant was constructed by homologous recombination. The function of srvg23535 was studied with comparing the colony morphology, motility, extracellular protease secretion, pressure sensing of H2O2 and Cu2+, iron utilization, antibiotic resistance, and metabolism between the wild type and the mutant strains. [Results] The biological characteristics analysis of srvg23535 knock-out mutant showed that the absence of srvg23535 had no significant effect on the colony morphology, motility, extracellular protease secretion, pressure sensing of H2O2 and Cu2+, iron utilization, antibiotic resistance and the metabolism of most of the tested carbon and nitrogen sources in V. alginolyticus. However, the mutant showed a weaker metabolism of D-trehalose, a stronger metabolism of pectin and alanine-glutamine (Ala-Gln), and accessibility to the utilization of alanine-aspartic acid (Ala-Asp) as the sole nitrogen source. [Conclusion] The sRNA srvg23535 is involved in the metabolism regulation of D-trehalose, pectin, Ala-Gln and Ala-Asp with laying the foundation for the target genes screening of srvg23535 and further elucidating the regulation mechanism of srvg23535 to its target genes.

Abstract:[Background] Proteases are widely used in the leather industry because the enzymatic dehairing approaches are environment-friendly. However, the instability to chemical reagents and the collagen degradation activity of proteases limit their industrial applications. [Objective] An alkaline protease gene from genome of Bacillus sp. N1 was cloned and expressed in Escherichia coli. The enzymatic properties of the recombinant protease were characterized and its application in dehairing process was discussed. [Methods] Genomic library was constructed to clone the protease gene aprG. The recombinant strain Escherichia coli BL21(DE3) pLysS/pET-28a-aprG was constructed for the protease expression with the induction of isopropyl β-D-thiogalactoside (IPTG). Characterization of the protease was performed through the forint phenol chromogenic method. The dehairing activity to the sheep and rabbit skin as well as feather of the protease was investigated. [Results] An alkaline protease gene aprG was cloned and expressed in E. coli expression system. The characterization of AprG showed that the optimum temperature and pH was 50 °C and 10.0, respectively. The metal ions showed soft influence on the AprG activity. Moreover, the protease AprG exhibited outstanding tolerance to surfactants, oxidants and reducing agents. The investigation on substrate specificity demonstrated that the protease displayed low hydrolysis ability toward collagen. The application study showed that AprG demonstrated effective dehairing on sheep and rabbit skin, and also showed high feather degradation activity. [Conclusion] The protease AprG has significant application potential in the leather industry.

Abstract:[Background] Escherichia coli is often used to produce threonine because of its excellent growth performance and clear genetic background. [Objective] In order to construct a high-yield strain of threonine, the nonessential genes of the E. coli THR in threonine biosynthetic pathway were knocked out while the key enzymes essential for threonine synthesis were heterologously expressed. [Methods] The lysC, pfkB and sstT of E. coli THR were deleted by using the FLP/FRT recombinant enzyme system. In addition, the recombinant plasmid with the genes lysCfbr and thrE from Corynebacterium glutamicum and the gene gapC from Clostridium acetobutylicum was constructed. Then this plasmid was introduced into different host strains. [Results] The target strain E. coli THR3 as obtained from the parental strain E. coli THR by deleting its threonine synthesis pathway: aspartate kinase III-coding gene lysC and phospho fructose kinase II-coding gene pfkB as well as the threonine absorption protein-coding gene sstT. The threonine production of strain E. coli THR3 as up to 75.64±0.35 g/L, was 9.9% higher than that of the parental stain E. coli THR (68.7 g/L). In addition, heterogonous expression of threonine secretory transporter-coding gene thrE and aspartic kinase-coding lysCfbr from C. glutamicum as well as heterologous expression of NADP+ dependent glyceraldehyde-3-phosphate dehydrogenase coding gapC from Clostridium acetobutylicum were beneficial to increase the threonine production. The recombinant strain E. coli THR6 could produce 105.3±0.5 g/L of threonine with a biomass of 18.26 g DCW/L and the threonine yield (g/g) from glucose increased by 43.20% and the acid production capacity per unit increased to 5.76 g/g DCW in fed-batch culture accumulation. [Conclusion] Knocking out a gene alone or modifying a pathway does not allow large amounts of threonine to be synthesized and accumulated. Co-transformation of multiple metabolic pathways is the most effective way to construct threonine engineered strains.

Abstract:[Background] Xilin river-riparian wetland-terrace grassland are the most representative aquatic-humid-terrestrial habitats in typical steppe region of Mongolia plateau, but little is known about the spatial characteristics of denitrifying bacterial communities in different habitats. [Objective] To reveal the composition, abundance, spatial distribution of denitrifying bacterial communities in different habitats of typical steppe region and associated spatial heterogeneity. [Methods] Using 454 pyrosequencing of 16S rRNA gene, we characterized community compositions and abundance of sediment/soil denitrifying bacterial communities from 6 sampling zones associated with aquatic, humid and terrestrial habitats. Based on the information of denitrifying bacterial 16S rRNA gene from previous references before 2014, we constructed denitrifying bacteria library as a reference to screen denitrifying bacterial genera associated with habitats. Meanwhile, we explored spatial heterogeneity of denitrifying bacterial genera by canonical correspondence analysis (CCA). [Results] The reference library contained 80 denitrifying bacterial species (65 genera). Accordingly, 36 denitrifying bacterial genera were screened out of total 469 bacterial genera from 6 sampling zones. Fourteen denitrifying bacterial genera co-existed in aquatic-humid-terrestrial habitats. Among them, Flavobacterium (1.65%?14.17%) and Hydrogenophaga (1.56%?1.69%) were dominant denitrifying bacteria across aquatic and humid habitats. Pseudomonas (1.85%) was the dominant bacteria only in low-floodplain wetland. The spatial distribution characteristics showed a tendency, first increased and then decreased along the aquatic-humid-terrestrial habitats, and reached peak value in low-floodplain wetland habitats. CCA analysis showed that Flavobacterium, Hydrogenophaga, Aeromonas and Sphingomonas were positively correlated with pH, water and sand contents. Whereas Bacillus, Streptomyces, Actinomadura were positively correlated with contents of clay, silt, organic matter and total nitrogen. [Conclusion] The compositions and abundance of denitrifying bacterial communities in typical steppe region showed obvious habitat heterogeneity. Low-floodplain wetland was the suitable habitat for growth and reproduction of denitrifying bacteria, which were driven by multiple environmental factors such as sediment/soil particle compositions, water content and pH.

Abstract:[Background] Yeast is a very important microorganism in wine fermentation. Its diversity and population composition play an important role in wine quality. There are many factors that affect the distribution and presence of yeast flora in wine. However, the influence of grape field management, for example the use of pesticides, on wine yeast community structure has not been reported yet. [Objective] We studied the effect of pesticide application on the community structure of wine yeasts during spontaneous fermentation. [Methods] Methods of pure culture, molecular biology identification and Illumina MiSeq metagenomic sequencing were used. [Results] In the spontaneous fermentation broth of grape samples without internal absorption chemical pesticides, 7 genuses (8 species) of yeast strains including Pichia, Hanseniaspora, Schizosaccharomyces, Candida, Saccharomyces, Zygoascus, and Issatchenkia were isolated and identified, while the result of metagenomic sequencing confirmed the dominance of Pichia (29.42%), Saccharomyces (21.91%), Issatchenkia (17.99%), Hanseniaspora (12.10%), Candida (7.47%), Zygosaccharomyces (5.32%), Schizosaccharomyces (3.07%), and Aureobasidium (0.29%). In the spontaneous fermentation broth of grape samples with conventional chemical pesticides, 5 genuses (6 species) of yeast strains including Pichia, Hanseniaspora, Schizosaccharomyces, Candida, and Cryptococcus were isolated and identified, while the result of Illumina MiSeq sequencing showed that Pichia (41.66%), Hanseniaspora (21.54%), Candida (19.11%), Zygosaccharomyces (7.78%), Schizosaccharomyces (4.04%), Cryptococcus (3.21%), Saccharomyces (1.12%), and Aureobasidium (0.49%) were involved in the fermentation. [Conclusion] Yeast community compositions in the two samples were significantly different, indicating that pesticide application on grapes had great influence on the community structure of wine yeasts during spontaneous fermentation.

Abstract:[Background] The nutritional value and flavor of feed ingredient was improved after fermentation and keratinase hydrolysis because microbiota compositions in the enzymolytic feed can be altered. In addition, enzymolytic soybean meal has significant improvements in growth performance and intestinal health. [Objective] Therefore, in this study, enzymolytic soybean meal (ESBM) was produced by fermentation and keratinase hydrolysis. The aim of this study was to explore the effect of two kinds of ESBM (keratinase hydrolyzed soybean meal (KHS) and keratinase hydrolyzed SBM with 30% inclusion of wheat bran (KHS-WB) on the microbiota diversity in the process of hydrolysis to drying. [Methods] First of all, the numbers of total bacteria, total mildew and yeast were measured by plate counting in laboratory. Additionally, Illumina MiSeq sequencing of 16S rRNA gene and ITS rDNA were employed to test the bacterial and fungal diversity dynamics of hydrolysis samples (0?36 h) in the pilot experiment. [Results] The plate counting result showed that the numbers of bacteria of two kinds of ESBM were 1 000?10 000 times higher than that of raw materials after treated with keratinase for 24 h. However, after drying, the counts of bacteria and fungi of ESBM were significantly decreased by 90%?99% than that of before drying. Moreover, the Illumina MiSeq sequencing results of 16S rRNA gene and ITS rDNA indicated that two kinds of ESBM had a similar alpha diversity index and microflora richness level. Additionally, the beta diversity of 16S rRNA sequencing result showed that Fructobacillus in KHS and Weissella in KHS-WB kept sustainable growth, occupied the privilege and became dominant bacterial genus (36 h), respectively. The results of ITS rDNA sequencing demonstrated that Aspergillus occupied the privilege in all genus of KHS while the diversity of dominant fungi was more abundant of KHS-WB in the process of hydrolysis. [Conclusion] Wheat bran as ESBM substrate can influence the microbiota diversity of ESBM, as well as the Weissella and yeast fungi will be the diversity of dominant microbes in the ESBM.

Abstract:[Background] Phytophthora capsici is a devastating soil-borne disease. Currently, it is mainly controlled by chemical fungicides, easily leading to environmental pollution and food contamination problems. [Objective] The aim is to isolate effective strains that can antagonize Phytophthora capsici. [Methods] Isolated strains were identified by the sequencing similarity of the internal transcribed spacer regions of rDNA and morphological characteristics. The antagonistic activity of the isolated strains was detected by the dual culture test. After treated with the extract from the isolated strains, malondialdehyde concentration, cellulase, β-glucosidase, and the polygalacturonase activity in Phytophthora capsaici were analyzed by the colorimetric method. [Results] All 11 isolated strains inhibited Phytophthora capsici growth. The isolated strains belonged to Trichoderma virens, Trichoderma harzianum, Trichoderma hamatum and Trichoderma asperellum species. Four strains (T. virens, T. harzianum, T. hamatum and T. virens) showed higher inhibition rates, which were 92.68%, 92.68%, 90.24% and 95.12%, respectively. After treated with the extract from the four isolated strains, the MDA concentration and the cellulase activity in P. capsici hyphae were significantly increased while the β-GC and PG activities were significantly decreased. The highest activity of chitinase was up to 0.831 U/mL with 0.783 U/mL more than that in control group. On the contrary, both of β-GC and PG activities were significantly decreased in the treated groups, in which the reduction rates ranged from 12.28% to 64.91% and 7.2% to 15.5%. [Conclusion] Four isolated Trichoderma spp. inhibited Phytophthora capsici growth by destroying its mycelial cytoderm structure, decreasing the activity of pathogenic factors and increasing the lipid peroxidation. This study provided a theoretical and technology support for resistant the Pepper Phytophthora blight.

Abstract:[Background] The crop diseases caused by Fusarium spp. are a frequent occurrence in Guizhou Province. As the climate characteristics of low temperature and high humidity in this region are not suitable for the mycelial growth of Fusarium pathogens, the infection and distribution of Fusarium have not been systematically investigated before. [Objective] To investigate the contamination of Fusarium spp. in the maize kernels collected from Guizhou Province. [Methods] Seventy-eight maize samples from 58 counties or county-level cities were collected and subjected to rapid immunoassay using prokaryotic expressed FvSG7-AP fusion protein that can be used to detect the presence of Fusarium pathogens in cereal grains. At the same time, biological culture tests were performed on some samples for further identification. [Results] A total of 67 samples were ascertained to be positive and the contamination rate was as high as 85.90%. The typical Fusarium mycelium and conidiospores were observed under microscope after biological culture. The Fusarium contaminated maize samples were distributed in 51 counties or county-level cities in Guizhou Province. Among them, mildly contaminated samples were detected in 19 regions, moderately contaminated samples were detected in 20 regions, and heavily contaminated samples were detected in 12 regions. [Conclusion] There are different degrees of Fusarium contamination in most parts of Guizhou. It is necessary to take effective prevention and control measures during the cultivation, harvesting and storage of crops to ensure food safety and human and animal health.

Abstract:[Background] In the process of postharvest, Phyllanthus emblica both as diet and medicine is easy to be perishable, which can seriously decrease its quality and economic value. [Objective] We identified the pathogen causing fruit rot of postharvest Phyllanthus emblica, its biological characteristics and cell wall degrading enzyme activity analysis, in order to provide the basis for rot disease control and extension of the storage life of Phylanthus emblica fruit. [Methods] The pathogen isolated from fruit rot of postharvest Phyllanthus emblica by tissue isolation method, followed by Koch’s postulates pathogenicity test. The pathogen was identified based on morphological characteristics and rDNA-ITS sequence analysis. In addition, the pathogenic mycelia growth and spore characteristics were determined, the activity of extracellular cell wall degrading enzyme activity were also detected. [Results] Thirty-two fungi isolates were isolated from the fruit rot of Phyllanthus emblica. Strain DQ23 was the causal pathogen fungus of fruit rot of postharvest Phyllanthus emblica. The pathogen strain DQ23 was identified as Penicillium choerospondiatis via morphological characteristics and rDNA-ITS sequence analysis. YDA medium was the most appropriate for mycelium growth. PSA was the optimum medium for conidia production. The mycelium could utilize a variety of carbon and nitrogen sources. For conidia production, the optimum carbon sources were sucrose and glucose, and the optimum nitrogen source were tryptone, beef extract and yeast extract. The best mycelium growth was obtained at 25 °C and pH 3.0?5.0. The conidia production increased rapidly at 25 °C and pH 4.0?7.0. Continuous light was beneficial to mycelial growth and spore production. Penicillium choerospondiatis could decompose pectin and cellulose, but not protein and tannin. [Conclusion] Penicillium choerospondiatis strain DQ23 was the pathogenic fungi causing fruit rot of Phyllanthus emblica, and possessed higher pectinase and cellulase activity.

Abstract:[Background] Colletotrichum gloeosporioides can infect rubber tree, and decrease rubber production. [Objective] An antagonistic strain SD-29 against C. gloeosporioides was isolated from farmland soil in Qingdao, Shandong province. The strain was identified, and its antifungal activity was evaluated. [Methods] The antifungal activity of strain SD-29 was determined using the methods of confrontation growth and mycelium growth rate. The crude extract of the fermentation broth was obtained by ethyl acetate extraction, and its activity was also evaluated. Then, strain SD-29 was identified based on morphology, physiological, biochemical characteristics and 16S rRNA sequence. [Results] Strain SD-29 had a strong inhibitory activity to C. gloeosporioides with the inhibition rate of 82.6%. The EC50 of fermentation broth extractions was 13.6 μg/mL. Crude extract (100 μg/mL) inhibited conidial germination with the inhibition rate of 63.16%, and its control effect on the rubber leaves infected with C. gloeosporioides was up to 48.96%. According to the results of morphology, physiological, biochemical characteristics and 16S rRNA sequence, strain SD-29 was identified as Streptomyces yatensis. [Conclusion] Strain SD-29 has a strong control effect on C. gloeosporioides.

Abstract:[Background] Mycoplasma synoviae (MS) can infect chickens and turkeys to cause airsacculitis, joint exudative, synovial bursitis and tendon sheath synovitis. Previous studies have shown that many metabolism-related enzymes in mycoplasma are present not only in the cytoplasm but also on the cell membrane surface, which act as adhesion associated proteins and play roles on the pathogenicity. The fructose-bisphosphate aldolases (FBAs) have been identified on the membrane and cytoplasmic fractions of Mycoplasma bovis and Mycoplasma gallisepticum, however, the MS FBA has not been studied before. [Objective] To explore the biological functions of metabolism related enzymes in MS, the FBA protein was subjected to bioinformatics analysis, prokaryotic expression, as well as immunogenicity and subcellular localization determinations in this study. [Methods] Using softwares of PSORTb, SignalP 4.1 Server and TMHMM Server to predict the subcellular localization, signal peptides and transmembrane regions of MSFBA. Blastn and MEGA 5.0 were used to analyze the homology and construct the phylogenetic tree of MSFBA. The full length of MS fba gene was amplified by overlap PCR and inserted into the expression vector of pET-28a(+). The recombinant MSFBA (rMSFBA) protein was then expressed in Escherichia coli BL21(DE3) and purified. The immunogenicity of rMSFBA was determined by Western blot analysis using MS-positive chicken serum. The rabbit anti-rMSFBA sera were prepared, and used to perform Western blot against the whole-cell, membrane and cytoplasmic fractions of MS to determine the subcellular localization. Suspension immunofluorescence assay further confirmed the non-membrane surface localization of the MSFBA. [Results] The MSFBA is predicted to be a cytoplasmic protein with no signal peptide and no transmembrane region. It is highly conserved protein which shows up to 99% homology among different MS strains, and 61% to 78% homology with different species of mycoplasma. The phylogenetic tree shows that the MSFBA is evolutionally closed to the FBA from Mycoplasma bovirhinis and Mycoplasma cricetuli, but remote to that of Mycoplasma arginini and Mycoplasma hominis. The rMSFBA protein was successfully expressed and purified, and the relative molecular mass was approximately 33 kD. The rMSFBA presented a specific binding to MS-positive chicken serum, which proved that it had good immunogenicity. Western blot analysis showed the rabbit anti-rMSFBA sera can react with the whole-cell and cytoplasmic fractions of MS bacteria, but not the membrane fraction, indicating the MSFBA protein is distributed in the cytoplasma. Suspension immunofluorescence assay further confirmed the FBA was not displayed on the membrane surface of MS cells. [Conclusion] The MSFBA protein is a highly conserved, immunogenic and cytoplasmic localized protein. The results provide a molecular basis for further study of the biological function of MSFBA protein.

Abstract:[Background] Mycoplasma synoviae (MS) is an important causative agent infecting chicken, which causes enormous losses to the poultry industry in China. [Objective] This study was designed to evaluate the pathogenicity of three isolates from MS outbreak farms, which could enrich our understanding about pathogenicity of MS isolates from different districts. [Methods] Systemic MS infection was induced experimentally in commercial broiler chickens with recent MS isolates (CHN-WF224-2016, CHN-BZJ2-2015 and CHN-JNB19-2016). The virulence of each strain was evaluated by detecting air sac and foot pad lesions, serologic response, tracheal mucosal thickness and MS isolation rates from the infected chicken at 10 and 21 days post-infection and comparing these results with those obtained from a live attenuated vaccine strain (MS-H) and uninfected controls. [Results] Isolates CHN-BZJ2-2015 and CHN-WF224-2016 induced foot pad lesions and typical infectious synovitis, especially CHN-BZJ2-2015 group. Serologic response and tracheal mucosal thickness measurements at 21 days post infection indicated that CHN-BZJ2-2015 isolate was significantly more virulent than the CHN-JNB19-2016 isolate and MS-H strain (P<0.05). [Conclusion] The variation in virulence of MS isolates emphasizes the importance of active, on-going control and prevention of MS in the chicken flocks of China and accumulates experimental data for vaccine development.

Abstract:[Background] Tarcoidosis in Trionyx sinensis occurred in a Trionyx sinensis farm in Liaocheng, Shandong, causing nodules in the viscera organs and limb muscles. Multiple infections occurred at the late stage of sarcoidosis and all viscera were stinky and rotten. According the survey, the infectious rate and mortality increased in 2017 compared with 2016. However, until now, no any report can be found on sarcoidosis in Trionyx sinensis in China. The route of infection, the pathogen, the method of prevention and control of sarcoidosis in Trionyx sinensis are unknown. [Objective] Our study aims to prevent sarcoidosis through analyzing the pathogeny of sarcoidosis in Trionyx sinensis and studying the pathematology. [Methods] According to disease diagnosis methods, separation and identification of pathogen were done after excluding parasites and virus infection. Meanwhile, in vitro sensitivity tests for pathogenic bacteria were used to screen sensitive drugs and to study the histopathology of the disease. [Results] Two dominant strains, DM1 and DM2, were isolated from the lesion and DM1 was the pathogenic bacterium of sarcoidosis in Trionyx sinensis according to regression infection tests. DM1 was Salmonella typhimurium through 16S rRNA gene sequencing and physiological-biochemical identification. The drug susceptibility analysis showed that pathogenic bacterium was sensitive to 14 antibiotics such as amikacin, gentamicin, cefoperazone, ciprofloxacin, and moderately sensitive to furazolidone and cephaloprim, but resistant to 18 antibiotics such as norfloxacin, rifampicin, ampicillin. Pathological section showed that the outer layer of the nodule was the capsule formed by fibrous connective tissues and the internal bean dregs like substance was eosinophilic coagulating necrosis. [Conclusion] Salmonella typhimurium was the pathogen of sarcoidosis in Trionyx sinensis and it can be prevented during the breeding process by feeding amikacin, gentamicin and similar antibiotics.

Abstract:[Background] Pig digestive tract disease is one of the most important diseases in the pig industry and brings certain economic losses to the swine industry. Escherichia coli is a common pathogen causing diarrhea of pigs at different ages, but mainly in young pigs. [Objective] The purpose of this study was to isolate and identify pathogens causing large-scale diarrhea in pigs on a large-scale pig farm in Meishan City, Sichuan Province. [Methods] Bacteria were isolated and identified from the infected pig liver, stomach and contaminated feed using conventional bacterial isolation methods combined with 16S rRNA gene sequence analysis. The isolates were tested for pathogenicity in mice, 16S rRNA gene phylogenetic analysis, virulence genes, and drug sensitivity testing. [Results] One pathogenic Escherichia coli was isolated from the liver of diarrhea pigs, one strain of Bacillus cereus was isolated from the stomach, and the source of infection was traced back to the feed on the farm. By detecting the corresponding virulence genes of these two strains, it was found that Escherichia coli is not extra-intestinal pathogenic type. Bacillus cereus was detected to have five virulence genes: nheA, nheB, nheC, bceT, entFM; Aminoglycosides and cephalosporins have good antibacterial effects on Escherichia coli. Erythromycin, florfenicol, cephalexin, cefoperazone have good antibacterial effect on Bacillus cereus, but the bacteria were not sensitive to conventional antibiotics such as penicillin and amoxicillin. [Conclusion] Feed is contaminated by Escherichia coli and Bacillus cereus.

Abstract:[Background] Plant endophytic fungi are an important source of natural bioactive substances. [Objective] The active substance of an antitumor endophytic fungus Aspergillus oryzae YX-5 isolated from Ginkgo biloba was investigated. [Methods] First, strain YX-5 was fermented. Then, the culture broth was extracted with ethyl acetate. By using vacuum liquid chromatography, Sephadex gel chromatography and high performance liquid chromatography, the active compound was isolated from the crude extract and the antitumor activity was measured by MTT assay. [Results] Four pure compounds were isolated from the crude extract of strain YX-5. Based on the analysis of NMR and HR-ESI-MS, these compounds were identified as hydroxyaspergillic acid (1), cyclo(4-Hydroxypro-Phe) (2), Cyclo(Leu-Phe) (3) and Cyclo(Ala-Phe) (4). Hydroxyaspergillic acid exhibited significant cytotoxic activity against HeLa cell with IC50 of 1.07 μg/mL. [Conclusion] Hydroxyaspergillic acid exhibited antitumor activity, indicating that Aspergillus oryzae and hydroxyaspergillic acid might have application potential in antitumor natural products development.

Abstract:[Background] Enterovirus 71 (EV71) is likely to cause severe hand, foot, and mouth disease (HFMD) that is sometimes associated with severe central nervous system complications, resulting in poor prognosis and even death of children. [Objective] To reveal the possible miRNA mechanism of EV71 increasing autophagy in human neuroblastoma cells, which might be related to EV71 induced neurological damage. [Methods] Western blot and qRT-PCR were used to demonstrate autophagy in EV71 infected SH-SY5Y cells. The differentially expressed miRNAs in SH-SY5Y cells with/without EV71 infection were detected by qPCR-Array. The functions of those miRNAs were analyzed by bioinformatics method. Then miRNA mimics was used to identify miRNAs associated with EV71-induced neuronal autophagy. [Results] EV71 could increase autophagy in SH-SY5Y cells and decrease the expression of miRNA29b in cells. Moreover, transfection of miRNA29b mimics in cells could not only reverse the down-regulated expression of miRNA29b induced by EV71, but also decrease the cellular autophagy induced by EV71 and the level of viral replication. [Conclusion] The results indicated that EV71 could induce autophagy in SH-SY5Y cells by down-regulating the expression of miRNA29b. Moreover, miRNA29b might be related to virus replication. Therefore, this study not only helps elucidate the specific molecular mechanism of neurological damage caused by EV71, but also lays a theoretical foundation for miRNA29b to become a new potential drug target for the treatment of EV71 infection.

Abstract:[Background] LncRNA-GAS5, a functional LncRNA that is encoded by Gas5 gene, plays an important role in the regulation of cell polarization, apoptosis, necrosis and autophagy. [Objective] To investigate the regulatory function of LncRNA-GAS5 on necrosis of macrophage RAW 264.7 induced by Bacillus Calmette-Guérin (BCG) infection. [Methods] The overexpression plasmid and interference plasmid of LncRNA-GAS5 were constructed and transfected into macrophage RAW264.7. Furthermore, the both transfected macrophage were infected by BCG. The cell viability was detected by MTT assay. The morphological changes of necrosis were observed with transmission electron microscope and the necrosis rate was analyzed by flow cytometry. The expression of RIP1, RIP3 and MLKL in mRNA and protein level were determined by qRT-PCR and Western blot. [Results] The expression of LncRNA-GAS5 in macrophage RAW264.7 significantly increased after BCG infection. The cell viability decreased and cell necrosis rate increased significantly when macrophage was transfected with the LncRNA-GAS5 overexpression plasmid alone or combined with BCG infection. Meanwhile, the mRNA and protein expression levels of RIP1, RIP3 and MLKL were upregulated dramatically and this process can be inhibited by LncRNA-GAS5 interference plasmid. [Conclusion] LncRNA-GAS5 can promote necrosis of RAW264.7 infected with BCG by up-regulating necrosis associated factors such as RIP1, RIP3, and MLKL. The results lay foundation on further insight into the molecular mechanisms of macrophage necrosis induced by BCG infection.

Abstract:[Background] Brown rot on peach and nectarine trees caused by Monilinia spp. is an important fungal disease, and produces serious economic losses frequently in China. There may exist more than one species of peach brown rot pathogens. [Objective] To clarify the species and pathogenicity of brown rot pathogens from different peach-growing areas in China. [Methods] The pathogens were isolated from peach brown rot fruits of different peach production areas, using the method of single spore isolation. Identification and analysis of isolates were carried out by morphological characteristics, molecular biology methods, and inoculation assay. [Results] In this study 15 brown rot pathogen isolates were obtained from 8 different peach-growing areas. The morphological characteristics of the isolates were slightly different from each other, but the isolate hfyn from Yunnan Province was significantly different from the other isolates. Based on its morphology, rDNA ITS sequence and phylogenetic analysis, the isolate hfyn was identified as Monilia yunnanensis, which was a newly found brown rot pathogen, and the other 14 isolates were identified as Monilinia fructicola. PCR amplification using several pairs of specific primers confirmed the morphological and rDNA ITS identification results. Inoculation tests showed that the pathogenicities on peach fruits of 15 isolates from different peach producing areas were different. The isolates from Lishui, Zhejiang Province, Shijiazhuang, Hebei Province, and Qingdao, Shandong Province had more virulent, and the virulences of isolates from Kunming, Yunnan Province and Tai’an, Shandong Province were relatively weak. [Conclusion] The results indicated that the major pathogen of brown rot in peach-growing areas in China was Monilinia fructicola, and there were differences in morphology and pathogenicity among these isolates from different peach-growing areas. The result from the present study may provide a scientific basis for managing peach brown rot epidemics.

Abstract:Biological nitrification plays a key role in the global nitrogen cycle, which is considered to consist of two serial steps of ammonia oxidation to nitrite biocatalyzed by ammonia oxidizing microorganisms (AOM) and nitrite to nitrate biocatalyzed by nitrite oxidizing bacteria (NOB). AOM include ammonia oxidizing bacteria (AOB) and ammonia oxidizing archaea (AOA), which are widely spread with relative abundances closely related to the concentration of ammonia. In 2015, three bacteria belonging to Nitrospira lineage II with NOB-specific functional genes were identified to carry AOM-specific functional enzymes including ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO), and proved their capacity to completely oxidize ammonia to nitrate. They have been named complete ammonia oxidizer (Comammox). Comammox could be subdivided into two branches of clade A and clade B based on the sequence discrepancy of ammonia monooxygenase subunit alpha (amoA), which have been widely found in natural and engineered environments such as soil (paddy soil, forest), fresh water (wetland, river, lake sediments, aquifers), wastewater and drinking water treatment plants. This paper introduces the discovery and distribution of Comammox, focuses on reviewing the research development on Comammox and prospects its research and application as a significant functional group of nitrogen cycle.

Abstract:Bacterial gene transcriptional regulation is one of the most widely studied mechanisms. Complex, but precise gene transcription regulatory network helps bacteria respond to environmental changes and plays a key role in the pathogenesis and transmission of pathogens. This paper discussed research progress of Yersinia pestis gene transcriptional regulation, the transcriptional regulation mechanism, strategies for transcriptional regulation, and the function of transcriptional regulation in Yersinia pestis pathogenesis and transmission, to provide new ideas for further research.

Abstract:Salt tolerance enzymes are active and stable at high salt concentrations and widely applied in high salt food processing, seafood processing, washing and other biotechnology fields with high salt concentrations. Salt tolerance genes can maintain normal function of microorganisms under high salt conditions. Therefore, obtaining and exploration of salt tolerance genes and salt tolerance enzymes in different environments have profound significance for revealing the salt tolerance mechanism of microorganisms and directing its application in the high salinity environment. The metagenomic technology skips the pure culture to explore the diversity and function of microorganisms and provide a new technology for discovering novel genes, developing new microbial active substances and studying microbial community structure and function. Combined with our own findings, this paper summarizes the strategies of using metagenomic technology to acquire salt tolerance enzymes and salt tolerance genes, and emphatically introduces the study of obtaining salt tolerance enzymes and salt tolerance genes from ocean, soil and gastrointestinal tract environment with metagenomics.

Abstract:The compounds derive from lipid metabolism in microalgae have important economic value, which can be used for the production of biofuels, nutraceuticals and green chemicals. Lipid metabolism processes are important in growth and development of microalgae and the response to stress. There are many similarities among microalgae, fungi and land plants in terms of lipid metabolism. With the recent advances in identification of functional genes related to lipid metabolism in microalgae, it has been found that lipid metabolism in microalgae have uncovered unique features, pointing out the necessity to analyze lipid metabolism in microalgae themselves. Chlamydomonas reinhardtii is a model organism to study lipid metabolism. Methods such as genom, transcriptom, proteom and metabolom are used to analyze lipid synthesis and lipid degradation processes in plastid, endoplasmic reticulum, and peroxisome. In this review, the authors summarized the recent research progress of lipid metabolism occurs in plastid, endoplasmic reticulum and peroxisome in Chlamydomonas reinhardtii.

Abstract:3-oxoacyl-acyl carrier protein reductase (FabG) are ubiquitously expressed enzymes that catalyse the reduction of acyl carrier protein (ACP) linked thioesters within the bacterial type II fatty acid synthesis (FASII) pathway, it reduce 3-oxoacyl-ACP to 3-hydroxyacyl-ACP, usually using NADPH as co-factor. FabG belongs to a broad superfamily of short-chain alcohol dehydrogenase/reductase or short-chain dehydrogenases/oxidoreductases (SDRs). The research reports of 3-oxoacyl-ACP reductase homologues in different bacteria show the characteristics of their diversity. However, in recent years, the summaries in this area have been very rare. In order to provide a theoretical reference for further research on 3-oxoacyl-ACP reductase, this review mainly summarizes the protein structures, function on fatty acid synthesis pathways and other biological functions of 3-oxoacyl-ACP reductase, antibiotics that target this enzyme.

Abstract:According to the training target of environmental engineering in the agricultural college, the course of Environmental Engineering Microbiology has been explored from the aspects of enriching teaching contents, discussing the key points of the course, improving teaching methods and innovating experiment contents. We paid the great attention to linking theory with practice. The scientific research and innovation ability of students were strengthened and the teaching quality was significantly improved.

Abstract:This project intends to adopt the presentation-assimilation-discussion (PAD) teaching method to carry out teaching reform exploration and practice in Food Microbiology, aiming to analyze the applicability of this method to Food Quality and Safety major courses in Medical universities. The results show that using PAD method in course of Food Microbiology improved the students’ enthusiasm for learning, capacity to think and explore, and skill to communicate. At the same time, it trained teachers’ organization ability, speech and evaluation skills. In addition, it improved the relationships between students and teachers and the relationships between students and students. The PAD method is simple and efficient for improving teaching quality.

Abstract:[Background] Pit mud quality has a decisive effect on the quality of Chinese strong-aromatype liquor. Acidogenic microbiota from pit mud significantly influence pit mud quality together with quality of the liquor. However, the evaluation criteria of pit mud quality are not very clear and accurate so far. [Objective] We aimed at establishing a quick and accurate method to evaluate pit mud quality in this study. Meanwhile, we analyzed functional microorganisms possibly participating in caproate production. [Methods] A cultivation method was developed to culture acidogenic microbiota. The concentration and ratio of subsequent metabolites were used to evaluate the caproate-producing capability of pit mud bacteria, as well as the corresponding pit mud quality. High-throughput sequencing technique was used to analyze the microbiota composition in the cultivation system. [Results] Compared to lactate and acetate, using glucose as the carbon source could effectively enrich caproate-producing microbiota in pit mud, resulting in higher caproate production. Small volume culturing and statistical sampling were determined. The concentration of caproate and the ratio of caproate to butyrate at the stationary phase of microbiota fermentation, could be served as accurate and stable indicators to evaluate pit mud quality. 16S rRNA gene sequencing showed that Uncultured bacterium Caproiciproducens were effectively enriched in the cultivation system. [Conclusion] Model system based on pit mud cultivation could be used to quickly evaluate pit mud quality in factories, and provides an approach to analyze caproate formation mechanism with pit mud microbiota.

Abstract:[Background] Colibacillosis and salmonellosis are the most common bacterial diseases in poultry, causing economically devastating to poultry industries. Salmonella and avian pathogenic E. coli (APEC) are important zoonotic pathogens, also with potential threat to human health. Thus, it is necessary to strengthen the rapid detection and differential diagnosis of these bacterial diseases. [Objective] We developed multiplex PCR to efficiently detect APEC, Salmonella enteritidis, Salmonella typhimurium, Salmonella gallinarum and Salmonella pullorum. [Methods] The special genes of APEC, Salmonella enteritidis, Salmonella typhimurium, Salmonella gallinarum and Salmonella pullorum were screened and selected for primers designing. A multiplex PCR method was developed by optimization of conditions. Then, the sensitivity and usage for detection of clinical isolates were determined. [Results] A multiplex PCR was established for accurately and effectively detection of APEC, Salmonella enteritidis, Salmonella typhimurium, Salmonella gallinarum and Salmonella pullorum. The sensitivity of the multiplex PCR was 103 bacterial colony forming units (CFUs) and 100 pg genomic DNA. The results of multiplex PCR for detection of clinical isolates agreed well with those of traditional serological assays. [Conclusion] Multiplex PCR developed in this study could efficiently detect APEC, Salmonella enteritidis, Salmonella typhimurium, Salmonella gallinarum and Salmonella pullorum.