Abstract:[Background] Quorum sensing refers to the phenomenon that bacteria use secreted signal molecules to communicate with each other, while quorum quenching refers to inhibiting the quorum sensing pathway by interfering with the production, release, accumulation or response of signal molecules. [Objective] To explore the diversity of quorum sensing and quorum quenching bacteria in sediment biofilm of Qingdao offshore. [Methods] Culturable bacteria were isolated from the sediment biofilm of Qingdao offshore by marine agar 2216E. The bacteria with quorum sensing and quorum quenching activities were screened by plate interaction and high-throughput screening methods. [Results] A total of 83 bacterial strains and 54 bacterial species were isolated, belonging to the four major phyla of bacteria: Proteobacteria, Bacteroidetes, Firmicutes and Actinobacteria. Among them, 38 strains (45.8%) produced acyl-homoserine lactones signal molecules (AHLs), belonging to Proteobacteria (37 strains, 15 species) and Bacteroidetes (1 strain, 1 species). The dominant bacteria belong to the genera Vibrio and Ruegeria. 57 strains (68.7%) can degrade AHLs, including Proteobacteria (41 strains, 23 species), Bacteroidetes (14 strains, 10 species), Firmicutes (5 strains, 5 species) and Actinobacteria (1 strain, 1 species), which showed high abundance and diversity. [Conclusion] The results of this study indicate that among the bacterial isolates from sediments biofilm in Qingdao offshore, quorum sensing and quorum quenching bacteria showed a high abundance and diversity. It laid a foundation for further research and development.

Abstract:[Background] The glycolytic housekeeping enzyme can be secreted to the extracellular matrix or located on the surface of the cell membrane, which plays an important role in the infection and cell adhesion of pathogen. The study of the secretion of glycolytic housekeeping enzymes in Edwardsiella, which is an important fish pathogen, is benefit to its pathogenesis and vaccine development. [Objective] To explore the secretion of housekeeping enzymes in glycolysis of Edwardsiella. [Methods] With ELISA, five housekeeping enzymes in the extracellular proteins from 48 different strains of of Edwardsiella was investigated. [Results] Five glycolytic enzymes are widespread in the extracellular protein of 48 strains of Edwardsiella. [Conclusion] Extracellular secretion of glycolytic enzymes in Edwardsiella is a common phenomenon.

Abstract:[Background] Epstein-Barr virus (EBV) is a common pathogen causing Burkitt’s lymphoma, Hodgkin’s disease, gastric cancer and nasopharyngeal carcinoma. A membrane protein BNLF2a encoded by EBV inhibits antigen transportation by transporter associated with antigen processing (TAP) and thereby evades the elimination by cytotoxic T cells. TAP is a member of the ATP-binding cassette (ABC) superfamily and composed of two subunits of TAP1 and TAP2. Using the energy of ATP hydrolysis, TAP transports antigenic peptides across the membrane with a conformational change. [Objective] The aim of this study was to study if BNLF2a affects the conformational switch of TAP. [Methods] Cysteines were introduced into the D-loop at the interface of TAP’s nucleotide binding domains. TAP were cross-linked by oxidizing agent Cu(II). The ratios of disulfide formed TAP were compared in the presence or absence of BNLF2a by western blot. [Results] The expression of BNLF2a increased the ratio of cross-linked TAP. [Conclusion] BNLF2a appears to stabilize TAP in the nucleotide binding domains dimerized conformation and thereby inhibit both of ATP and antigenic peptide binding to TAP.

Abstract:[Background] It is now well accepted that low frequency electromagnetic field (LF-EMF) would produce a variety of health effects. [Objective] The objective of this work is to further investigate the effects of LF-EMF radiation on cells’ protein expression and enzymes activities. [Methods] Saccharomyces cerevisiae was used as the research object and was continuously exposed to 50 Hz low frequency electromagnetic field in this work, then the expression of its intracellular proteins and relevant enzyme activities were analyzed. [Results] The effect of LF-EMF treatment on the activities of SOD (superoxide dismutase), CAT (catalase) and MDH (malate dehydrogenase) in S. cerevisiae appeared a trend of firstly promoted and then inhibited. LF-EMF treatment can increase 43.18% of the activity of SOD at 72 h, 12.22% of the CAT at 16 h, and 12.90% of the MDH at 20 h (P<0.05); however, no significant effects of LF-EMF treatment on the activity of ADH (alcohol dehydrogenase) and LDH (lactate dehydrogenase) were observed (P>0.05). In addition, the results of protein electrophoresis and proteomics analysis indicated that LF-EMF could change the expression level of intracellular proteins in S. cerevisiae, including significant up-regulation of 3 proteins and significant down-regulation of 9 proteins (P<0.05). [Conclusion] LF-EMF radiation can affect the activity of relevant enzymes in S. cerevisiae, and change the expression level of some intracellular proteins.

Abstract:[Background] Aureobasidium pullulans belongs to “black yeast” and can synthesize melanin. A. pullulans displays characteristic polymorphic cell types, such as yeast-like cell (YL), swollen cell (SC), chlamydospore (CH), hyphae (HY), monilioid hyphae (MH), septate swollen cell (SSC), meristematic structure (MS). The swollen cell can be further differentiated into chlamydospore, monilioid hyphae, septate swollen cell and meristematic structure. The differentiation of A. pullulans can be regulated by various factors such as pH, temperature and nutritional conditions. [Objective] To study the effects of different oxygen density, temperature, osmotic pressure, pH value and nutrition level on the morphology of A. pullulan cells. [Methods] The microscopic techniques and methylene blue staining were used to observe the effects of different conditions on cell morphology of A. pullulans. [Results] Growth of A. pullulan was not observed under completely anaerobic condition. Under high oxygen condition, the yeast-like cells budded at the early nutrient-rich stage. However, once the nutrient was gradually used up, the yeast-like cells started to form swollen cell and then to chlamydospore. Under low oxygen condition, the yeast-like cell grew into HY through SC at the beginning of incubation when nutrients are rich. The different nutrient concentration had significant effects on the polymorphic differentiation of A. pullulans. Yeast-like cells were the main types of cells in the yeast extract peptone dextrose medium (YPD) medium with high nutrition and optimal ambient conditions. The YL cell differentiated into SC or HY to adapt to or escape the environment in the potato dextrose agar (PDA) medium. The malt extract agar (MEA) medium was nutrient deficient, therefore the SC or HY differentiated into dormant cell CH or MH. The 10% NaCl depressed the growth rate of A. pullulans and inhibited the synthesis of melanin. The effect of 10% KCl or 10% Na2SO4 on the cell differentiation of YL was the same as 10% NaCl, which suggests osmotic stress prevented the differentiation of yeast-like cells into hypha or chlamydospore. The SC was more resistant to high temperature than YL, but less resistant than MS. [Conclusion] Nutrition levels are the key factor regulating cell morphogenesis of A. pullulans.

Abstract:[Background] The Haematococcus pluvialis is the best source of natural astaxanthin and is widely used in industrial production of astaxanthin. [Objective] The current study is to explore influences of different concentrations butylated hydroxytoluene (BHT) on accumulation of astaxanthin in H. pluvialis, with a view to build the BHT technology system for enhancing astaxanthin content of H. pluvialis. [Methods] Without nitrate bold’s basal medium was selected as the induction medium for the culture of Haematococcus pluvialis strain LUGU. Meanwhile, high light treatment medium maintained induction conditions. the effects of different concentrations of butylated hydroxytoluene (BHT) on the growth, astaxanthin accumulation, reactive oxygen species, antioxidant system and the expression of related gene were studied in Haematococcus pluvialis under high light and nitrogen deficiency. [Results] The results showed that the astaxanthin content was enhanced efficiently in alga cells treated with 2mg/L BHT, within the range of 0–3 mg/L BHT, reaching 31.66 mg/g. In addition, 2 mg/L BHT treatment group effectively reduces the level of reactive oxygen species, increases intracellular nitric oxide levels, improves the algal cells catalase (CAT), peroxidase (POD), superoxide dismutase (SOD) activity and glutathione (GSH) content, inducing efficient expression of lcy and chy. [Conclusion] Under abiotic stress conditions, exogenous BHT could promote astaxanthin accumulation, which was associated with BHT mediating the levels of ROS and NO, and astaxanthin biosynthetic related genes expressions in H. pluvialis.

Abstract:[Background] The enrichment and culture technology based on the nitrifying bacterial community show the efficiency and stability in removing ammonia nitrogen, nitrite nitrogen and other harmful nitrogen from the aquaculture water. However, only a few studies are available on nitrifying functional strains for the targeted cultivation of nitrifying bacterial community in aquaculture. [Objective] To study the effects of salinity, pH, temperature and ventilation on the growth and nitrification activity of strain XH1 isolated from nitrifying bacterial community. [Methods] Different gradients of salinity, pH, temperature and ventilation were assayed. The growth of strain XH1 and the corresponding removal effect on ammonia and nitrite nitrogen were analyzed by counting the number of bacteria and measuring the concentrations of ammonia and nitrite nitrogen, respectively. [Results] Strain XH1 could grow well under the conditions of salinity 5‰?35‰, pH 6.0?9.0, temperature 15?45 °C and ventilation 0.5?1 V/(V·min). The maximum content of bacteria reached the level of 2.34×109 cells/mL. Under the condition of salinity 5‰?35‰, pH 6.0?9.0, temperature 15?30 °C, and ventilation capacity 0.5 V/(V·min), the removal efficiency of ammonia nitrogen was significant (P<0.05). The highest removal rate of ammonia nitrogen in the culture solution could reach 86%?97% on 1?3 d, but then the concentration of ammonia nitrogen increased slightly in the following days. In addition, the highest removal rate of nitrite nitrogen was up to 68%. [Conclusion] The strain XH1 had a good adaptability to major environmental factors such as salinity, pH, and temperature. With the excellent removal ability on ammonia nitrogen, strain XH1 could be used as an alternative strain for the agents’ research on harmful nitrogen prevention and control in medium-low-salinity aquaculture ponds.

Abstract:[Background] Powdery mildew (PM) is one of the most devasting diseases in rose and other ornamental plants, however, there is no better way to control it untill now. Previous studies have demonstrated that endophytes are beneficial to host plant’s disease resistance. Rosa multiflora and R. multiflora var. carnea is a PM resistant and susceptible rose species, respectively. It’s not clear about the difference of their fungal endophytic communities and their function. [Objective] To study the difference of the fungal endophytic communities between two rose species and to understand the possible role of endophytes on PM resistance. [Methods] The culturable endophytic fungi (EF) were isolated from two rose species at different developing stage of PM by traditional EF isolation methods. [Results] A total of 2?003 endophytic fungi were isolated from 2 880 tissue segments of 24 samples of two rose species, of which 1 333 from R. multiflora and 670 from R. multiflora var. carnea. The isolates were identified to 30?taxa according to the morphological charateristics and the rDNA internal transcribed spacer (ITS) analysis, of which Alternaria alternata, C. truncatum and Phomopsis amygdali were the dominant genera of two rose species at different developing stage of PM. Contrary to this, Seimatosporium sp., Coprinellus sp. and Chaetomium globosum etc. were only found in R. multiflora or R. multiflora var. carnea. [Conclusion] The fungal endophytic community of R. multiflora showed significantly difference from that of R. multiflora var. carnea. The average colonization rate (CR) of EF from R. multiflora was significantly higher than that of R. multiflora var. carnea at each developing stage of PW (P<0.05, Chi-square test). The role of the specific and dominant EF of R. multiflora in PM resistance were further studied in the future.

Abstract:[Background] Xingyun Lake is one of plateau lakes of Yunnan. The study of its diversity of yeasts and the screening of carotenoids-producing strains contribute to the development and utilization of the yeast resources of Xingyun Lake. [Objective] To study the diversity of community structure of yeasts in Xingyun Lake, and to screen the carotenoids-producing strains. [Methods] Yeast separation was carried to on the water samples of Xingyun Lake by membrane filtration flat culture. Identification was based on sequence analysis of D1/D2 domains of 26S rRNA gene, and to combinate with morphological characteristics and physiological and biochemical indicators, and the diversity of yeast was analyzed by software BIO-DAP. The carotenoids were extracted by acid-heating method, and the content of carotenoids were determined by ultraviolet spectrophotometer. [Results] A total of 797 strains were isolated from the water samples of Xingyun Lake, and identified as 18 genera and 37 species. The Rhodotorula mucilaginosa and Naganishia albida are the dominant species, accounting for 36.64% and 28.86% of the total strains, respectively. There were 28 strains of yeast carotenoids with a yield of more than 200 μg/g cell dry weight, and 4 strains of carotenoeds-producing were up to 300 μg/g cell dry weight: Rhodotorula glutinis (2 strains), Rhodotorula mucilaginosa (1 strain) and Rhodosporidiobolus fluviale (1 strain). [Conclusion] The yeasts of Xingyun Lake are rich in diversity, and the degree of lake nutrition and geographical difference affect the structure of yeast community. In Xingyun Lake, Rhodosporidiobolus and Rhodotorula are the major groups of carotenoids-producting, and carotenoids-producting were up to 300 μg/g of cell dry weight. It has the value of further development research.

Abstract:[Background] Transglutaminase (TGase) is an enzyme that catalyzes the acyl transfer reaction, which can catalyze cross-linking reactions between or within various protein molecules, and has important potential value in the food, cosmetics, medicine and other fields. [Objective] Molecular engineering and site-directed mutagenesis were carried out by cloning glutamyl transferase from Streptoverticillium ladakanum B1 to obtain efficient heterologous expression in E. coli. [Methods] The gene of pro and TG from Streptoverticillium ladakanum were cloned into pET-22b to generate co-expression and fusion expression, respectively. Based on these two expression patterns, the first four amino acids of the mature TGase N-terminal were modified by site-directed mutagenesis to detect the effect of different expression patterns and mutation on enzyme activity. [Results] When co-expression of the pro-peptide with TGase, the active form of TGase can be obtained directly, and the specific activity reaches 37.71 U/mg. Based on the fusion expression, the first three amino acid DSD of the N-terminal of TGase were mutated to AAA, and the specific enzyme activity reached 14.04 U/mg, which was 14.05% higher than the original expression pattern. [Conclusion] The co-expression of pro-peptide with TGase can directly produce the active form of TGase. For the fusion expression pattern, the mutation of the appropriate site is beneficial to increase the TGase activity.

Abstract:[Background] Subtilisin is an important industrial protease with many applications. [Objective] The aim of this study is to improve the subtilisin production by screening promoters and signal peptides, and optimization of fermentation medium. [Methods] Bacillus licheniformis BL10, constructed in our previous research, was served as the host strain for subtilisin production, and four promoters (PbacA, P43, PaprE and PsrfA) and four signal peptides (SPVpr, SPSacB, SPSacC and SPAprE) were screened to improve subtilisin production, and the medium for subtilisin production was further optimized. [Results] Based on our results, the expression ratios among these four promoters were PbacA>PaprE>P43>PsrfA, and the secretion efficiencies were SPAprE>SPSacC>SPSacB>SPVpr, respectively. Among these strains, BL10/pPbacA-aprE displayed with the highest subtilisin activity (275.21 U/mL), which was increased by 64% compared with that of BL10/pHY-aprE (167.98 U/mL). Furthermore, the fermentation medium was optimized, and the orthogonal test was further applied to enhance subtilisin production. The maximum subtilisin activity (747.37 U/mL) was obtained in the optimized medium with 40.0 g/L corn starch, 50.0 g/L soybean meal, 4.0 g/L (NH4)2SO4, 3.0 g/L K2HPO4, 1.0 g/L CaCO3, increased by 4.45-fold compared with that of the initial condition. [Conclusion] Collectively, this study provided an effective strategy for enhanced production of subtilisin.

Abstract:[Background] Nitrogen fertilizer input for production in China is very high, which is one of the most serious impediments of the sustainable development of agriculture. Research on biological nitrogen fixation (BNF) has increased significantly because of its potential importance to economy and the environment. [Objective] The aim of this work was to screen and identify efficient endophytic nitrogen-fixing bacteria of sugarcane, and to demonstrate their potential for associative nitrogen fixation with sugarcane as well as plant growth promotion. [Methods] An endophytic diazotroph strain NN08200 was isolated from the stem of sugarcane, and its nitrogen fixation was tested further by using the acetylene reduction assay and PCR amplification of the nifH gene encoding the iron protein of nitrogenase. Identification of the strain species according to its culture character and the microscopic observation, automatic bacteria identification system and by analyzing their 16S rRNA gene sequences. To demonstrate its potential for associative nitrogen fixation with sugarcane and growth promotion activities, strain NN08200 was inoculated into micropropagated sugarcane seedlings to investigate their plant growth-promoting and associative BNF activities using the 15N isotope dilution technique. [Results] The acetylene reduction activity of strain NN08200 was 2 445 nmol C2H4/(h·mL), and PCR amplification of nifH amplified approximately 339 bp fragments from the strain. Cloning and sequencing of the amplicon showed that strain NN08200 contained sequences that were the closest match to nifH. Based on phenotypic characteristics, Biolog system test, and 16S rRNA gene sequence analysis, strain NN08200 was identified as Pantoea sp. Compared with the uninoculated controls, strain NN08200 significantly promoted the growth of micropropagated sugarcane seedlings by increasing dry weight and plant height of sugarcane. 15N isotope dilution assays demonstrated that the associative nitrogen fixation rates of NN08200 in the sugarcane roots, stems and leaves were 7.49%, 15.02% and 10.79%, respectively, which were higher than that of Gluconacetobacter diazotrophicus strain PAL5, whose associative nitrogen fixation rates in the sugarcane roots, stems and leaves were 3.53%, 9.44% and 4.87%, respectively. [Conclusion] Strain NN08200 has the potential to efficiently fix N2 in sugarcane and promote sugarcane growth.

Abstract:[Background] Previous results showed that the DDT-degrading strain, Chryseobacterium sp. PYR2, could efficiently remove DDT and other pollutants from contaminated soil and was great potential in bioremediation. However, the influence of PYR2 on plants and also microorganism-plant interactions is not clear. [Objective] In this study, the plant growth-promoting efficiency and its mechanism of strain PYR2 was investigated, thus providing the theoretical basis for the further development of DDT degradation and plant growth-promoting dual-effect bacterial agents. [Methods] Seed soaking and plant pot experiments were conducted to reveal the plant growth promoting effects of PYR2 on wheat. Salkowski method was used to detect the concentration of indole-3-acetic acid (IAA). Single factor experiments were performed to detail the effects of different culture conditions on its growth and IAA yield. Liquid chromatography-tandem mass spectrometry-multiple reaction monitoring (LC-MS/MS-MRM) detection was employed to detect the intermediate metabolites of IAA biosynthetic process. [Results] Seed germination rates of wheat was greatly enhanced, and so the growth of wheat plants by using cell suspension of PYR2, as the significant increase of height, lateral root number, fresh weight as well as dry weight. The optimum IAA synthesis conditions were temperature 30 °C, pH 7.0?8.0, salt ion concentration <0.5%, L-tryptophan 50 mg/L. The intermediate metabolites of IAA synthesis were tryptophol (TOL), tryptamine (TAM) and indole-3-acetamide (IAM), suggesting that there are three different IAA synthetic pathways in PYR2 (IPyA, TAM and IAM). [Conclusion] Strain PYR2 exhibited significant plant growth promoting effects on wheat, due to its multiple and effective IAA biosynthetic pathways. These results suggest that PYR2 is greatly potential in the bioremediation of and plant-growth in pesticide contaminated soil.

Abstract:[Background] Sugarcane smut disease is a main disease on sugarcane that causes considerable yield losses; rhamnolipids are biosurfactants that have been studied as an antifungal agent against various of plant diseases. [Objective] To evaluate the antifungal activity of rhamnolipids against S. scitamineum in vitro, exploring the preliminary antifungal mechanism of rhamnolipids against S. scitamineum. [Methods] The teliospores of sugarcane smut germination were employed to determine the antifungal of rhamnolipids. The method of mycelial growth rate and dry weight of mycelium were used to test in in vitro test of rhamnolipid. The effect of rhamnolipids on the membrane permeability of S. scitamineum that was assayed the change of electrical conductivity of the hyphae. [Results] The results showed that rhamnolipids could significantly inhibit the spore germination of S. scitamineum. 2.0 g/L rhamnolipids reduced the germination rate of teliospores by 45.03%. Rhamnolipids significantly inhibited the mycelial growth of the diploid, haploid-a and haploid-b strain of S. scitamineum. Rhamnolipids could increase the membrane permeability of cell membrane of S. scitamineum, compared with the control, the conductivity of diploid strain raised 9-fold at 2.0 g/L rhamnolipids after 5 min，the conductivity of haploid-a strain of S. scitamineum increased by 94.23% after 30 min at 2.0 g/L rhamnolipids. The conductivity of mycelium increased by 54.49% while haploid-b strain of S. scitamineum had been handled with 0.1 g/L rhamnolipids for 30 min. Mycelial conductivity raised with the increasing of the concentration of rhamnolipids. [Conclusion] Rhamnolipids showed a high antifungal activity against S. scitamineum, which could offer a new method for controlling sugarcane smut disease.

Abstract:[Background] The intestinal microbes are the main driving force of crayfish to degrade cellulose and hemicellulose. [Objective] The study of the relative abundance of bacteria in the intestine provides theoretical support for revealing the role of intestinal microorganisms in the degradation of lignocellulose. [Methods] The diversity of intestinal bacteria was studied by Illumina MiSeq high-throughput sequencing, and xylanase production bacteria were isolated from crayfish intestine by plate screening method. [Results] Morphological and 16S rRNA gene molecular identification showed that all 4 strains belonged to Bacillus genus. The four strains were identified combining the physiological and biochemical characteristics. The results showed that the strain Z-3 was identified as Bacillus subtilis, strain Z-4 was Bacillus velezensis, strain Z-29 was Bacillus cereus, strain Z-30 was Bacillus altitudinis. The results of 16S rRNA gene high-throughput sequencing showed that: at the genus level, the intestinal bacteria of crayfish were mainly Candidatus Bacilloplasma, Bacteroides, Vibrio, Acinetobacter, Dysgonomonas, Tyzzerella 3, Aeromonas and Shewanella. [Conclusion] Crayfish had abundant bacterial resources in the intestine, and Bacillus bacteria played an important role in the degradation of lignocellulose.

Abstract:[Background] High-temperature stacking is a key step among the whole sesame-flavor Baijiu fermentation process, and vital for the formation of typical flavor of sesame-flavor Baijiu. [Objective] This study aimed to analyze prokaryotic and eukaryotic microbial communities during the high-temperature stacking process. [Methods] Illumina high-throughput sequencing was performed on the bacterial 16S rDNA V3–V4 region and the fungal ITS2 region of the high-temperature stacked Jiupei samples. The change pattern of prokaryotic and eukaryotic microbial community structure were analyzed. The changes in temperature, moisture, acidity, starch and reduced-sugar during stacking were also analyzed. [Results] At the beginning of 0–6 hours, the dominant bacteria were Bacillus, Weissella, and Acetobacter. After 20 h of stacking, the flora changed significantly. Weissella decreased and the Acetobacter increased. At the beginning, the dominant fungi were Aspergillus, Saccharomyces, and Pichia. After 6 hours, the proportion of Aspergillus dropped sharply, while Pichia and Saccharomyces gradually increased, and their absolute dominance maintained until the end of the stacking. The overall structure of the microbial community was temporarily affected by the turn-over operation (when it was piled up for 20 h), eventually returning to equilibrium. After 36 h of stacking, both prokaryotic and eukaryotic microbial community stabilized. Reduced-sugar data showed that the saccharification was rapid in the early stage, later became stable after 36 h. Temperature, acidity and starch content were also stable at this stage. [Conclusion] During the high-temperature stacking-fermentation process, fluctuation patterns of the prokaryotic and eukaryotic microbial community structure were different. The prokaryotic community changes were based on the turn-up at 20 h. The dominant species before the turn-up were Bacillus, Weissella and Acetobacte, the dominant strains after the turn-up were Weissella and Acetobacter, the latter was absolute dominant. Compared to prokaryotic community changes, the eukaryotic microbial community concentration gradually, by the end of the stacking, Pichia and Saccharomyces accounted for more than 93% of the total eukaryotic microbial. During the high-temperature stacking of the sesame-flavor Baijiu, the propagation of various prokaryotic/eukaryotic microbes and the corresponding changes of microbial communities laid the microbial basis for later high-temperature fermentation.

Abstract:[Background] Lentinula edodes virus disease is important for L. edodes production. Previous to this study, L. edodes mycovirus HKB (LeV-HKB) and L. edodes partitivirus 1 (LePV1) were proved to be the two major mycoviruses identified in L. edodes germplasm, and co-infection of these two viruses was also prevalent. [Objective] The purpose of this study was to develop a quick and efficient technique for detection of these two viruses in the early stage of L. edodes production. [Methods] Three pairs of specific primers for LeV-HKB, LePV1 and actin genes (β-actin) of L. edodes were designed based on the reported sequences, respectively. The crucial factors of multiplex RT-PCR including template concentration, primers concentration, dNTPs concentration, annealing temperature, and number of cycles were then optimized. [Results] A multiplex RT-PCR detection method for LeV-HKB (or LeV-HKB related virus) and LePV1 was developed. [Conclusion] The multiplex RT-PCR assay developed in this study had high specificity and repeatability using the virus detection of L. edodes core collections.

Abstract:[Background] “Replacing wood by grass” is a concept to use other substrates than wood for edible fungi cultivation, to effectively alleviate the contradiction between edible fungi and forests. [Objective] To optimize a “replacing wood by grass” substrate formula by using different agricultural residues to replace sawdust. [Methods] In accordance to simplex lattice design method, 7 different agro-residues, namely wheat straw, corn straw, soybean straw, peanut straw, rape straw, rice straw and corncob were used for Pleurotus djamor cultivation. Enzyme activity of cellulase, polyphenol oxidase and laccase in the growth process of the mycelium was measured. The formulation suitable for the growth of Pleurotus djamor was analyzed and optimized. [Results] Soybean straw could significantly increase the mycelium growth of Pleurotus djamor, followed by wheat straw. Polyphenol oxidase activity of rape straw was highest. The optimized high mycelium growth formula includes (%, W/W), wheat straw 55.3, soybean straw 19.7, wheat bran 15.0, corn flour 5.0, bean pulp 3.0, lime 1.0 and gypsum 1.0. [Conclusion] A suitable substrate composed of various agricultural wastes instead of sawdust for mycelial growth of Pleurotus djamor was optimized for “replacing wood by grass” strategy.

Abstract:[Background] Animals harbor many microbial species that exert profound effects on host physiology and pathology, currently emerging as one of investigational hotspots. [Objective] To explore Staphylococcus epidermidis effects on the developmental timing of Drosophila melanogaster. [Methods] The bacteria were isolated from the fly guts by carotenoid expression medium (CEM), and the bacterial species were further determined by using 16S rRNA gene sequence. The growth and development of flies were detected. Real-time quantitative PCR was used to detect the activation of the prothoracicotropic hormone (PTTH) and insulin pathway in flies. [Results] The isolated strain was identified as S. epidermis, which was able to effectively colonize in the guts of flies. S. epidermis accelerated the development of D. melanogaster by promoting the growth rate. At the molecular level, S. epidermis activated PTTH and insulin signals to stimulate the growth of hosts. [Conclusion] S. epidermis was a commensal bacterium of flies, which stimulated the growth and development of flies by regulating PTTH and insulin signals.

Abstract:[Background] Diseases, especially bacterial diseases have been a big challenge in American bullfrog (Rana catesbiana) farming. Due to a high diversity of these pathogens, rapid spread of the disease as well as high mortality rate, the bacterial disease in the bullfrog is difficult to prevention and control. [Objective] In order to study the taxonomic status and pathogenicity, one bacterial strain NW1203 was isolated and identified from diseased R. catesbiana. [Methods] The strain NW1203 was isolated from R. catesbiana by streaking plate method under aseptic conditions, followed by morphological observation, physiological, biochemical tests and 16S rRNA gene phylogenetic analyses to identify bacterial species. To investigate the pathogenicity of these bacteria, artificial infection followed by hemolytic test and histopathological examination were performed. [Results] Based on the characterization of physiological, biochemical examination and 16S rRNA gene sequence alignment, the isolated bacteria NW1203 was identified as Chryseobacterium sp. with 100% sequences similarities to Chryseobacterium sp. F30. Furthermore, phylogenetic trees showed bacteria NW1203 was grouped with Chryseobacterium sp. Hemolytic tests showed the strain NW1203 exhibited strongly hemolytic to the erythrocyte of sheep, mice and bullfrog. Artificial infection test and tissue section observation of infected frogs indicated the strain NW1203 had strongly pathogenic to R. catesbiana with LD50 values of 4.753×103 CFU/g and could cause severe pathological changes in main tissues including liver, kidney and spleen. [Conclusion] This study indicates that the bacteria strain NW1203 was a new pathogen causing bacterial disease of R. catesbiana and provides a theoretical basis for the prevention and control of bullfrog diseases.

Abstract:[Background] Due to the special physiological structure of ruminants, the previous researchers focused on the structure and composition of the rumen microbes, which seriously ignored the important role of cecum microorganism in nutrient digestion and intestinal health. [Objective] The study was designed to reveal the bacterial diversity in cecum and their community structure from goat using MiSeq sequencing technology. [Methods] Twelve 10-month-old and weight of 20.70±1.60 kg goats were selected for collecting cecum liquid after normal feeding, after 20 d, the cecum fluid from each goat was collected and then total genomic DNA was extracted from the cecum fluids. High variable region of bacterial 16S rRNA genes was amplified by PCR using bacterial universal primers. Analyzing amplicon sequence data on the Illumina MiSeq sequencing platform and biological information analysis using QIIME software were done. [Results] In this sequence, 813 496 effective sequences and 6 883 OTU were obtained, and according to the rarefaction curve and coverage index showed that the result of the sequencing was more comprehensive covering the cecum microorganism community of goats. Alpha diversity and beta diversity analysis showed that there were differences in cecum microbial diversity among goats. At the phylum level, the dominant bacteria were Firmicutes and Bacteroidetes, and the core genus was composed of Clostridium, Ruminococcus and unclassified bacteria of six. The cecum microbes of goats are mainly metabolizing function, including carbohydrate metabolism, amino acid metabolism, energy metabolism and lipid metabolism. [Conclusion] The diversity of bacteria in the cecum and rumen of goats was significantly different, similar to the composition of fecal microorganisms, and the composition of cecum microorganisms in both goats and single-gastric animals had similarities and differences.

Abstract:[Background] The membrane protein (M) of porcine epidemic diarrhea virus (PEDV) plays an important role in the viral assembly process, membrane fusion and viral replication, but the mechanism of interaction between M protein and host cells is still unclear. [Objective] Co-immunoprecipitation technique coupled with LC-MS/MS were used to screen cellular proteins interacting with PEDV M protein, which can provide a foundation for revealing the function of M in viral multiplication. [Methods] PEDV DR13 vaccine strain was inoculated into monolayer of Vero cells at a MOI of 0.1. After 36 hours of infection, the cells were collected and lysed. Host cellular proteins that interact with the M protein of PEDV were immunoprecipitated using the M monoclonal antibody, then identified by LC-MS/MS, and analyzed by gene ontology (GO) annotation. Among them, two interested cellular proteins were further confirmed with Co-IP and cellular colocalization. [Results] Based on the analysis of the number of peptide segments, 218 cellular proteins interacting with M protein were identified. These host cellular proteins are closely related to protein synthesis, metabolism and cell signaling pathway transduction. Cell division cycle 42 (CDC42) and eukaryotic translation initiation factor 3 subunit L (eIF3L) were chosen for reverse Co-IP reconfirmation and colocalization analysis. The results showed that both CDC42 and eIF3L protein interact with M protein. [Conclusion] This study identified that PEDV M protein could interact with CDC42 and eIF3L proteins in host cells, and identified 60 other host proteins that might interact with M protein. The study is to provide an important theoretical basis for the study of the interaction between PEDV and host cell proteins.

Abstract:[Background] Melanin has many biological activities such as anti-tumor and anti-radiation, which make it of great application potential in production and life. Fungi is an important source of melanin, and it has shorter production cycle and higher yield compared with animals and plants, which make it easy to achieve commercial application. [Objective] The application, development and innovation trend of fungal melanin were revealed to provide references for researchers and enterprises that dedicated to fungal melanin industry. [Methods] Based on IncoPat scientific and technological innovation platform, we searched and counted the related patents of fungal melanin around the world, and made comprehend analyses of them from the aspects of involved strains, the composition of patent technology, and patent values of the applicants. [Results] The results of analysis showed that the number of patent applications for production and preparation was the largest in the field of fungal melanin (50.56%), while the number of applications as chemical dyes and cosmetics was smallest (13.48%). The main strains involved the following genus: Fomes, Aureobasidium, Auricularia, Inonotus, Lachnum, Ganoderma and Aspergillus. At present, the hot technical fields of patent applications of fungal melanin are mainly C12 (microbial fermentation; medium; mutations or genetic engineering) and A61 (medicine), and we predict that both of them will continue to be hot fields of technical applications for quite a while to come; China has the largest number of fungal melanin patents, but the percentage of high-value patents was relatively low, and there is still a certain gap compared with foreign countries. [Conclusion] Chinese researchers need to enhance creative application and overseas layout of core patent technologies in medicine and chemical industry, strengthen cooperative research with enterprises and technology transfer, in order to seize the application market of fungal melanin in these fields, and promote the development of fungal melanin related patents related to a higher quality.

Abstract:[Background] Metacordyceps neogunnii was a kind of new resource with the potential application value because it possessed multiple pharmacological functions. However, more active compounds were still worthy of exploring. The research of its active compounds mainly focused on macromolecular or polar compounds up to date, few studies were paid to micromolecular or weak-polar compounds. [Objective] The non-polar or weak-polar small molecular compounds of the petroleum ether extracts from M. neogunnii were researched in this study, in order to improve the chemical fingerprint database of the active substances in the M. neogunnii. [Methods] M. neogunnii were extracted with petroleum ether by soxhlet extraction. The substance components were identified and analyzed by fourier transform infrared spectrometry (FTIR) and gas chromatography-mass spectrometry (GC-MS). [Results] According to FTIR, the analyses found that the components of extracts included the functional groups such as C?H, C=O, C?O and C=C; Through GC-MS method, 109 compounds including alkanes, arenes, olefins, acids, esters, alcohols, amines and others were identified. The non-polar or weak-polar small molecular compounds such as steroids, aromatics, alkanes, amides, olefins and phenols were discovered for the first time; And the relative contents of linoleic acid and its isomer were the highest, up to 38.33%. [Conclusion] Many small active components from M. neogunnii were obtained by petroleum ether extraction, which supplemented the chemical composition and provided scientific data for high value-added utilization of M. neogunnii.

Abstract:[Background] The prevalence of neuroAIDS and Cryptococcus neoformans meningitis has significantly increased and the mechanism remains unclear. [Objective] To study the inhibitory effect of SBi4211 on adhesion of C. neoformans to human brain microvascular endothelial cells (HBMEC) induced by HIV-1 gp41. [Methods] The adhesion experiments were carried out to determine whether SBi4211 could block C. neoformans adhesion. Furthermore, the expression of hyaluronic acid CD44 was analyzed by the Western Blot method (WB) during the adhesion process. [Results] SBi4211could significantly inhibit the HIV-1 gp41-enhanced adhesion of C. neoformans to HBMEC in a dose- and time-dependent manner (P<0.05). Meanwhile expression of the C. neoformans hyaluronic acid receptor CD44 on HBMEC is down regulated. [Conclusion] Our study suggested SBi4211 could block HIV-1 gp41-enhanced adhesion of C. neoformans to HBMEC by reducing expression of receptor CD44, which might provide novel insights into understanding the mechanisms of C. neoformans and HIV-1 associated comorbidity besides its prevention and treatment.

Abstract:[Background] Klebsiella pneumoniae is a ubiquitous opportunistic bacterium. The high prevalence of extended-spectrum beta-lactamases (ESBLs) in Klebsiella pneumoniae has propelled the need to explore alternative antibacterial therapies. [Objective] The present study focused on the isolation and characterization of a novel virulent Klebsiella pneumoniae extended-spectrum beta-lactamases-producing phage isolated from sewage therapy. Moreover, we used the mouse model of septicemia to examine the efficacy of phage therapy in treating infections caused by K. pneumoniae produced ESBLs. [Methods] The morphology of the Klebsiella phage F20 was observed with electron microscopy. One-step growth kinetics, host range, and pharmacokinetics of the phage were determined. In additional, F20 was used to rescue mice from bacteremia caused by extended-spectrum beta-lactamases-producing Klebsiella pneumoniae KP-20 in vivo experiments. [Results] F20 exhibited lytic activity evident in clear areas on the bacterial lawns. Morphologically, F20 was classified as a member of the Siphoviridae family and the Caudovirales order. The phage is highly infectious with a short latent period (18 min) and a large burst size (89 PFU/cell). The phage is stable over a wide pH range (5.0 to 9.0) and at high temperatures (50 °C). Administration of F20 after KP-20 challenge significantly decreased the bacterial burden in the blood and organs (lung, liver, spleen and kidney) of mice, and bacterial titers significantly decreased by 1?3 orders of magnitude in mice treated with phage therapy (P<0.001). Administration of F20 can rescue 87.5% of the mice and have no adverse effect, but the control group resulted in a 0 survival rate within 1 day. Phage treatment can significantly improve the survival of mice (P<0.001). [Conclusion] Our study provides the experimental evidence that F20 shows significant treatment efficacy against Klebsiella pneumoniae infection in mice without any adverse effects. The characteristic of the phage F20 greatly increase its utility as biological bactericide.

Abstract:The oqxAB is a multidrug-resistant efflux gene encoding the OqxAB efflux pump. It comprises two open reading frames, oqxA and oqxB, and confers bacterial resistance, especially to quinolones. OqxAB, discovered in E. coli plasmid pOLA52 is one of the important members of plasmid-mediated quinolone resistance (PMQR) genes. Until now, 11 oqxA and 32 oqxB alleles have been found respectively. OqxAB can be disseminated horizontally through the transmissible plasmid and to increases the resistance level of the receptor, resulting in the more difficult treatment. Although the gene is currently mainly prevalent in Enterobacteriaceae, it cannot be excluded in non-Enterobacteriaceae bacteria, which will pose a huge threat to human and livestock health. This review summarizes the discovery, drug resistance mechanism and epidemiological characteristics of the oqxAB and its alleles, aiming to provide literature data for the rational use of quinolone in clinical treatment and the process of food animal production, as well as reduce the prevalence of oqxAB.

Abstract:Microorganisms play an important role in tobacco fermentation and aging. In this review, we address the role of microorganisms in tobacco fermentation and aging, including the distribution of microorganisms on tobacco, their application and research progress. The division of microorganism on tobacco, the dynamic change of microorganism during the process of fermentation and aging and the application of inoculating microorganism are mainly introduced. Some research and application results showed that tobacco leaf microorganisms could shorten the period of fermentation and aging, improve the quality of flue-cured tobacco, reduce harmful substances and improve smoking safety. Finally, the subsequent research of correlation studies is also prospected.

Abstract:The bifunctional aldehyde/ethanol dehydrogenase (AdhE) exhibits acetaldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH) activities, and is one of the key enzymes in the bacterial ethanol anaerobic fermentation pathway. Researches on bacteria-host interactions revealed that AdhE also plays an important role in bacteria adapting to the internal environment changes and regulating bacterial virulence. In this paper, we summarized the regulatory mechanisms of AdhE on bacterial pathogenesis and host immune, which may provide new perspectives for understanding AdhE’s function.

Abstract:The genus of Massilia was proposed by La Scola et al with Massilia timonae UR/MT95T as the type species in 1998. In the last decade, studies on this genus have got much attention and undergone fast developments. Up to date, this genus comprises 43 validly described species, which belongs to the family Oxalobacteraceae of the class Betaproteobacteria in the phylum Proteobacteria. Members of genus Massilia widely distribute in soil, as well as clinical specimen, plant, water, ice core, air and the surface of rock. Massilia can not only synthesize multiple secondary metabolites and enzymes, but also have many functions such as phosphorus solubilization, degradation of phenanthrene and resistance to heavy metals. Massilia has attracted the attention of researchers in various countries. This paper reviews the research progress of the establishment of Massilia, taxonomic characteristics, their distribution in the environments, as well as the application prospect in soil remediation, chemical and medical industry.

Abstract:[Background] Due to the serious public safety problem caused by the propagation of pathogenic microorganisms, such as Ebola virus, the governments of all countries pay much attention to laboratory biosafety. [Objective] To assess the safety status of pathogenic microorganisms in Chinese universities will benefit for the specific-targeted laboratory management measures. [Methods] Based on literature review and interview with some experts of biosafety management, we designed the questionnaire and the investigation was carried out with the participation of 341 technicians and students from 50 Chinese universities. [Results] Some important steps for management of pathogenic microorganisms were obvious insufficient, especially in biosafety education, establishment of safety management system, construction of standardized laboratory, biological waste disposal, and laboratory safety facilities maintenance. [Conclusion] The universities should put safety construction responsibility into effect so as to eliminate the above latent dangers of safety hazards.