Abstract:[Background] The strains in this study were respectively isolated from the wild edible fungus fruit-bodies of Lepista irina, L. panaeolus and L. nuda, which were collected from Guancenshan and Wulushan Mountains in Shanxi province. [Objective] To obtain their optimal culture conditions on mycelial growth. [Methods] The effects of different carbon sources, nitrogen sources, carbon-nitrogen ratio, pH and culture temperature were studied by using mycelial growth rate as an indicator; according to the Box-Benhnken center combination based on the experimental design principle, the three-factor and three-level response surface method is used to determine the optimal culture carbon source, nitrogen source and pH for the mycelium. [Results] The mycelium growth rate of L. irina reached 1.13 mm/d under the optimized culture conditions of glucose 20.9 g/L, potato 196.47 g/L, pH 6.0 and culture temperature 21 °C; the maximized mycelium growth rate of L. panaeolus was 0.73 mm/d under mannitol 17.4 g/L, yeast extract 8.1 g/L, vitamin B 0.1 g/L, K2HPO4 2.5 g/L, MgSO4 2.5 g/L, pH 7.9, 25 °C; L. nuda in potato 200 g/L, soluble starch 20.5 g/L, KNO3 2.1 g/L, K2HPO4 2.5 g/L, MgSO4 2.5 g/L, vitamins B 0.1 g/L, pH 7.0, 25 °C reached the maximum mycelial growth rate of 2.38 mm/d. [Conclusion] the optimized culture conditions for three strains of Lepista will provided the data for the introduction and acclimatization of wild edible fungi.

Abstract:[Background] Tobacco flavor is an important auxiliary material in cigarette production. Microbial contamination usually occurs in the processes of transportation and storage of tobacco flavor, especially the residual casing, which could cause spoilage of tobacco flavor. [Objective] To investigate the diversity of spoilage microorganisms in the deteriorated tobacco flavor and identify the main spoilage microorganisms causing the deterioration of tobacco flavor. [Methods] Dilution plate counting method was applied to monitor the microbial composition of the deteriorated tobacco flavor from Yunnan, Fujian and Guangxi of typical hot-humid areas in China, and then the 16S rRNA, ITS or 26S rRNA genes of the isolates were sequenced. Furthermore, verifiable experiments were used to identify the main spoilage microorganisms causing deterioration of the tobacco flavor. [Results] The tobacco flavor samples from the three typical hot-humid areas in China were with different degree of bacteria, molds or yeasts contamination, and the bacterial contamination was the most common. A total of 76 bacterial strains, 9 fungal strains and 42 yeast strains were isolated. The 76 bacterial strains were distributed in 2 phyla, 4 classes, 5 orders, 10 families and 15 genera. Ralstonia, Citrobacter, Enterobacter and Bacillus were the dominant genera. The 9 fungal strains were distributed in 3 classes and 6 genera of the Ascomycota phyla, with 1?2 fungal strains belonging to each genus. The 42 yeast strains were distributed in 6 genera of Ascomycota. Pichia, Wickerhamomyces and Saccharomyces were the dominant genera. The bacterial composition of Yunnan and Fujian samples was obviously more than that in Guangxi. The fungal composition of Guangxi and yeasts’ composition of Fujian were more than those in the other two sites, respectively. The verifiable experiment results showed Citrobacter, Enterobacter, Bacillus, Ralstonia and Pichia were the main spoilage microorganisms in the tobacco flavor samples. [Conclusion] The study on the composition of contaminated microorganisms and the confirmation of spoilage microorganisms in the deteriorated tobacco flavors could help to control the contamination of microorganisms and the spoilage of tobacco flavors in hot-humid areas in China.

Abstract:[Background] In addition to orchid mycorrhizal fungi, the roots of orchids harbor plant fungal endophytes termed root-associated fungi. [Objective] Three endangered photosynthetic Cypripedium species distributed in coniferous forest and shrub habitats were screened for root-associated fungi using culture-dependent (isolations from root fragments) techniques. The species richness and the degree of root-associated fungi community differentiation of examined Cypripedium species and two different habitats were determined. Ecological function analysis of root-associated fungi was also estimated. [Methods] RAF were isolated from surface sterilized root fragments of orchids. Total DNA were extracted from isolated root-associated fungi, and internal transcribed spacer (ITS) regions were amplified. The ITS-PCR products were sequenced. Phylogenetic analysis was applied. Species richness and diversity of fungal communities of studied orchid species and collecting sites were estimated. After blasting the ITS sequences of root-associated fungi in NCBI database, the annotation of the closest matched sequences were used to analyse the ecological function. [Results] 278 root-associated fungi isolates, corresponding to 25?operational taxonomic units (otus), were identified, including 23 Ascomycota OTUs and 2?Mucoromycota OTUs. The root-associated fungi species richness of Cypripedium tibeticum was higher than that of Cypripedium flavum. The degree of root-associated fungi community differentiation of different Cypripedium species is bigger than that of different habitats. Fungal taxa in the roots of the three Cypripedium species could be assigned to 3 trophic modes, which were symbiotroph, saprotroph and pathotroph, respectively; and 8 guilds, which were ectomycorrhizal, plant pathogen, endophyte, animal pathogen, fungal parasite, ericoid mycorrhizal, undefined saprotroph and uncertain, respectively. [Conclusion] This study revealed the distribution characteristics and ecological function of RAF in roots of three Cypripedium species sampled from two different habitats, and also laid a foundation for the symbiotic relationship study of root-associated fungi and Cypripedium species in the future.

Abstract:[Background] Anoxygenic phototrophic bacteria (APB) have been widely investigated and applied in the bioremediation of nitrogen-polluted aquaculture. Various organic compounds are predominated in the contaminated aquaculture water, among them, organic nitrides significantly hamper the nitrogen removal efficiency. [Objective] In order to investigate the effects of organic compounds and salinity on inorganic nitrogen removal (ammonia, nitrate and nitrite) and elucidate the mechanism of inorganic nitrogen removal by Rhodobacter azotoformans YLK20, moreover, further to develop well-adapted microbial agents with high nitrogen removal efficiency. [Methods] RAST and KEGG analyses were used to elucidate carbon and nitrogen metabolic pathways and salt-tolerant mechanism(s) of YLK20. The removals of ammonia, nitrate and nitrite were measured by sodium hypobromite oxidation, UV spectrophotometry and N-(1-naphtyl)-ethylenediamine dihydrochloride spectrophoto-metric method, respectively. [Results] Genomic analysis revealed that YLK20 possesses EMP, HMP, TCA, nitrogen fixation, ammonium assimilation and ammonification and denitrification pathways. It contains several salt-tolerant genes such as sohB, nhaC, betB and gbsA. Pyruvate, acetate, citrate, ethanol and mannitol stimulated the cell growth and the nitrogen removal of YLK20, while glucose and fructose decreased the nitrogen removal level. Nitrate and nitrite were removed efficiently in the presence of sucrose while it is difficult to remove ammonia. In the presence of high concentration of peptone (3.21 g/L) and urea (1.43 g/L), inorganic nitrogen concentrations were significantly decreased. YLK20 was able to tolerate to at least 3% of NaCl, efficiently remove the inorganic nitrogen under low salinity conditions while nitrite removal was significantly inhibited in the presence of the high salinity. The inorganic nitrogen both from freshwater and marine rearing water could be efficiently removed by YLK20. [Conclusion] YLK20 could efficiently remove the inorganic nitrogen by ammonium assimilation and denitrification pathways, especially in the presence of high concentration of organics nitrogen. YLK20 was tolerant of high salinity, which is suitable for both freshwater aquaculture and marine aquaculture. YLK20 as a specific microecological modulator, was capable of removing efficiently nitrogen, showing the great potentials in the application in aquaculture remediation.

Abstract:[Background] Apart from symbiotic fungi and algae, the lichens symbiosis system also includes some symbiotic actinomycetes. [Objective] To collect lichen samples from Xishuangbanna, Baimang Snow Mountain of Yunnan and South Bank of the Baltic Sea in Germany, and to analyze the diversity of pure cultivate actinomycetes associated with lichens. [Methods] Three actinomycetes selective media were used to isolate actinomycetes through plate dilution method. The taxonomic status of pure cultured actinomycetes was determined by comparing 16S rRNA gene sequence similarity and constructing phylogenetic tree. [Results] A total of 1 123 strains were isolated and 417 strains were identified. Among them, 107 strains of actinomycetes, including 18 potential new taxa, were isolated and purified from 17 lichen samples of Xishuangbanna which distributed in 7 orders, 14 families and 33?genera. Streptomyces was distributed widely. One hundred and three actinomycetes were separated from 7 lichen samples of Baimang Snow Mountain which belonging to 4 orders, 5 families and 9 genera. Sixteen possible new species were acquired. Streptomyces was the dominant genus, accounting for 39% of the total number of the actinomycetes. Total 65 strains of actinomycetes were obtained from 5 lichen samples of South Bank of the Baltic Sea which distributed among 4 orders, 8 families and 18 genera. Five potential new taxa and Streptomyces were the prime group. [Conclusion] In this study, the diversity of actinomycetes associated with lichens in Xishuangbanna was more abundant than Baimang Snow Mountain and South Bank of the Baltic Sea. Members of streptomycetes were predominant, and potential new taxa accounting for 15.5% of total actinomycetes associated with lichens in Baimang Snow Mountain. The floristic composition of actinomycetes associated with lichens in the three regions was different, which was closely related to the geographical environment and completely different climate of the three regions.

Abstract:[Background] Heterotrophic nitrifying-aerobic denitrifying microorganisms have received more and more attention, because they achieve the vision of simultaneous nitrification and denitrification in one system. The nitrogen removal pathway is different along with different bacteria. While the nitrogen metabolic pathway of bacteria is direct correlation with the type and activity of nitrogen elimination enzyme. So find out the nitrogen removal pathway of Pseudomonas alcaliphila AD-28 will provide technical support in application. [Objective] In order to reveal the nitrogen removal mechanism, the nitrogen removal characteristic and key enzymes for nitrogen degradation of Pseudomonas alcaliphila AD-28 were studied. [Methods] The nitrogen removal characteristic of strain AD-28 was investigated when sodium citrate was used as carbon source, ammonium sulfate, sodium nitrite and potassium nitrate were used as nitrogen sources. At the same time, the activities of key enzymes-ammonia monooxygenase (AMO), hydroxylamine oxidoreductase (HAO), nitrite reductase (NIR) and nitrate reductase (NAR) were measured. [Results] After 24 h treatment by strain AD-28, bacteria density (OD600) reached 1.971, the degradation rates of NH4+-N, NO3?-N, NO2?-N and total nitrogen (TN) were all exceeded 96% when the initial concentrations were 18.85, 26.13, 19.47, 66.11 mg/L, respectively. The corresponding specific activities of AMO, HAO, NIR and NAR were 0.028, 0.003, 0.011, 0.027 U/mg, respectively. [Conclusion] The results suggest that AD-28 is a simultaneous heterotrophic nitrification-aerobic denitrification strain. The nitrogen removal pathway of the stain was deduced as following. NH4+-N was oxidized to NH2OH by AMO, then NH2OH oxidized to NO2?-N by HAO, NO2?-N and NO3?-N removed from the medium by NIR and NAR.

Abstract:[Background] In recent years, due to the mining and smelting of metal ores, the processing and use of arsenic products, the burning of coal and other factors, arsenic pollution in the soil environment has become more and more serious, causing many people to be exposed to extremely dangerous arsenic poisoning. [Objective] To study the diversity of endophytic bacteria aoxB gene in Pteris vittata root of Wanshun Pb-Zn mining area in Sichuan, and provide a theoretical basis for improving the efficiency of soil heavy metal pollution ecological restoration. [Methods] Real-time quantitative PCR (qPCR) and restriction fragment length polymorphism (RFLP) were used to study the abundance and diversity of arsenic oxidation gene (aoxB) of the endophytic bacteria in Pteris vittata root which were isolated from a Pb-Zn mining area in Hanyuan of Sichuan province, China. [Results] The results of QPCR showed that there was a significant difference in the expression of aoxB genes between the different sampling sites. The abundance aoxB gene were in the order of dressing area>entrance to the mountain>spoil area>mine tailings>mine mouth. The RFLP results showed that there were significant differences in the diversity index of the aoxB gene of endophytic bacteria among different sampling sites, and the diversity index of the species were in the order of mine tailings>mine mouth>spoil area>outside mine area>mine mouth. Pearson correlation analysis showed that the abundance of aoxB genes was negatively correlated with concentration of As (p<0.05), and the diversity index was positively correlated with concentration of Pb and As (p<0.01). Phylogenetic analysis showed that aoxB containing endophytic bacteria in the Pteris vittata were mainly belonged to Alphaproteobacteria. [Conclusion] The results showed that there were abundant endophytic bacterial populations containing aoxB gene in Pteris vittata root, and these endophytic bacteria showed potential application value.

Abstract:[Background] Halophilic microorganisms living in hypersaline environment, and conceiving unique physiological and metabolic characteristics, are one type of important microbial resources from extreme environment. [Objective] To uncover species diversity of cultivable halophilic microorganisms in athalassohaline salt mines in China, and to accumulate strains of halophilic microorganisms, this research was conducted. [Methods] Culture-dependent approach was applied to analyze the halophilic microorganisms from salt core samples of Dingyuan salt mine, Anhui province, China. Classification of the culturable halophilic microorganisms was performed based on 16S rRNA gene sequencing and sequence similarity search. On this basis, some representative strains were subjected to colony morphology, salt tolerance and enzyme activity assay. [Results] In total, 264 strains were obtained via culture-dependent approach, of which 150 strains were classified as halophilic archaea, accounting for 56.8%, and 114 strains were affiliated to halophilic bacteria, accounting for 43.2%. Results of 16S rRNA gene sequence similarity search showed that those strains belonged to six haloarchaeal genera, i.e. Halorubrum, Halopenitus, Haloterrigena, Natrinema, Natronoarchaeum and Natronomonas, and five bacterial genera, i.e. Pseudomonas, Aliifodidinibius, Halobacillus, Halomonas and Halospina. Through the enzyme activity assay, one strain producing extracellular protease, one strain conceiving esterase activity and two strains presenting amylase activity, two strains hydrolyzing the gelatin, were found among these selected species. Furthermore, species diversity index of the halophilic archaea was higher than that of halophilic bacteria. [Conclusion] This study reported the species diversity of the culturable halophilic microorganisms isolated from the Dingyuan athalassohaline salt mine (Anhui, China). This study gives us a better understanding of the microbial resources in athalassohaline salt mines, and accumulates rich microbial resources for further application.

Abstract:[Background] Class Ⅱ lantibiotics are ribosomally synthesized and post-translationally modified peptides, mainly produced by Gram-positive bacteria. In the last step of its biosynthesis, the N-terminal peptidase domain of transporter protein LanT cleaves the leader peptide to produce active lantibiotics. However, the removal mechanism of leader peptide in this class lantibiotics is still not clear. [Objective] To investigate the effects of cleavage sites on the activity of peptidases domain BovT150 and SboT150 from different streptococci. [Methods] Expression vectors for the precursor peptides with mutated cleavage sites were constructed by site-directed ligase-independent mutagenesis and then the wild-type precursors (BovAm and SboAm), their mutant precursors, as well as the corresponding peptidases (BovT150 and SboT150) were expressed and purified in E. coli. The precursors were separately incubated with each peptidase in vitro and the removal efficiencies of the leader peptides were assessed by HPLC, antimicrobial activity assay and MALDI-TOF MS. [Results] Both cleavage sites GG and GA of BovAm and SboAm allowed BovT150 to retain peptidase activity, and Gly was more suitable to be processed by BovT150. Only cleavage sites GG and GA of SboAm were accessible to SboT150, which cleaved Ala more efficiently. [Conclusion] The change of amino acid residues at the cleavage sites of leader peptides affected the efficiencies of class Ⅱ lantibiotic peptidases in varying degrees.

Abstract:[Background] Chrysosporium was a kind of important keratinophilic fungi. [Objective] The Chrysosporium resources in soil samples from Gansu and Yunnan provinces were investigated. [Methods] Different soil samples were collected, and cultivated with the chicken feather and hairs by baiting technique, then the target strains were isolated and identified by the morphological characteristics and phylogeny of ITS rDNA sequences. [Results] There were five target strains of Chrysosporium obtained, strain H5.11 was very similar to the original description of C. articulatum in morphology and clustered into a subclade with the sequence of C. articulatum from GenBank in phylogenetic tree; Strain H10.10 was consistent with C. georgiae in morphology, both of them appeared red colony, and it was closely related to the type sequence of C. georgiae in phylogeny; Strains EB8803M and EB8801M had similar morphological characters to C. submersum, and it clustered together with sequences of C. submersum from GenBank in phylogenetic tree; Strain O1 was similar to the original description of C. zonatum in morphology and it also clustered together with the sequence of C. zonatum from GenBank. Therefore, these strains were identified to four species, C. articulatum, C. georgiae, C. submersum and C. zonatum. [Conclusion] These four species are new to China.

Abstract:[Background] Carbamoyl phosphate is an important precursor in metabolism of arginine and pyrimidine, and plays a key role in biosynthesis of arginine and pyrimidine and its derivatives in microorganisms. [Objective] The synthesis of carbamoyl phosphate through different pathways was investigated and compared in Escherichia coli BW25113. [Methods] Ornithine carbamoyltransferase (OTC) was overexpressed in E. coli BW25113 as a carbamoyl phosphate detecting system. Then carbamate kinase (CK) from different species or carbamoyl phosphate synthase II (CPS II) was overexpressed in E. coli BW25113 to enhance carbamoyl phosphate biosynthesis. These two carbamoyl phosphate biosynthetic pathways (CK pathway and CPS II pathway) were then optimized by adjusting substrates supply and introducing L-glutamine synthetase. [Results] A carbamoyl phosphate detection system was established in vivo by overexpressing OTC in E. coli. By overexpressing CKs from different species in E. coli with OTC, the carbamoyl phosphate biosynthetic pathway was enhanced, and the results showed that CK from E. coli was better than the other two, which produced 2.95±0.15 mmol/L L-citrulline in 9 h whole-cell bioconversion. By overexpressing CPS II in E. coli with OTC, the carbamoyl phosphate biosynthesis was improved, and 3.16±0.29 mmol/L L-citrulline was obtained in the whole-cell biocatalytic process. The CK and CPS II pathways were optimized by changing the concentration of substrate NH4HCO3 to 100 mmol/L and introducing exogenous L-glutamine synthetase (GS). As a result, 4.67±0.55 mmol/L and 6.12±0.38 mmol/L L-citrulline was achieved by strains with CK or CPS II pathway respectively. After the optimization of CPS II pathway with overexpressing GS, extra addition of L-glutamine was not required in the reaction system. [Conclusion] Biosynthesis of carbamoyl phosphate was improved by constructing and optimizing the CK synthetic pathway or CPS II synthetic pathway. CPS II pathway with introducing GS is better than that of the CK synthetic pathway in respect of carbamoyl phosphate synthesis, and provides a more efficient strategy for the synthesis of arginine, pyrimidine and its derivatives.

Abstract:[Background] The large-scale agricultural reclamation and development of oasis ecosystem in Xinjiang have ecological impacts on the environment. Thus, it is important to evaluate the effect of human activities on the soil biological characters, especially the soil bacterial community, to provide a guidance for rational utilization of land and sustainable development of agricultural resources in the future. [Objective] By comparing soil bacterial structure and diversity in cotton fields affected by agricultural reclamation, with those in poplar forests where ecological environment has largely been kept intact, we studied the effects of human activities on agricultural reclamation and development on soil microbial ecology. [Methods] The national standard method was used to measure the soil physical and chemical properties, including total nitrogen, total salt, organic matter, alkaline hydrolysis nitrogen, available phosphorus, available potassium and pH in the cotton fields and poplar forests in the lower and middle reaches of Tarim river basin. Total soil DNA was extracted, a library was established by PCR amplification, and 16S rRNA gene (V4 region) was high-throughput sequenced using Illumina Hiseq 2500 sequencing technology platform. Bioinformatics was used to analyze the changes of bacterial α-diversity (such as Chao1, observed species, phylogenetic diversity (PD), Shannon, Simpson and Good’s coverage indices) and β-diversity in the two areas, and redundancy analysis was used to find the main factors that may have affected the changes of the bacterial community structure in the both areas, as well as community function prediction results were also used to give an auxiliary interpretation. [Results] Basic physical and chemical properties of soil analysis: compared with the primary poplar forest in oasis cotton field, there were significant increases in total nitrogen, alkaline hydrolysis nitrogen, salt and pH, and significant positive correlation between soil salt content and total nitrogen, alkaline hydrolysis nitrogen and pH (P<0.05). Bacterial diversity analysis: the diversity index of chao1, observed species and PD in cotton field samples were significantly lower than those in poplar forest samples. The Shannon index difference was not significant, but it showed the same variation trend with the above three indexes. Bacterial community composition analysis: the dominant bacteria across the two places were Alphaproteobacteria, Actinomycetes, Gemmatimonadetes, Bacteroides, Acidobacteria, Gammaproteobacteria, Betaproteobacteria, Deltaproteobacteria, Chloroflexi, Firmicutes, Planctomycetes, Crenarchaeota and Nitrospirae (>1%). According to the results of linear discriminant analysis, the significant enriched microorganisms in cotton field soil were Acidobacteria, Actinobacteria, Chloroflexi, Firmicutes, Flavobacteria and some species of Alphaproteobacteria, Gammaproteobacteria, and one species (Candidatus Nitrososphaera) belonged to Crenarchaeota was also discovered in cotton field. However, in original poplar forest soil samples, the significant enriched microorganisms were Chlorobi, and Deltaproteobacteria. Some species in Proteobacteria and the Nitrosopumilus which belonged to Crenarchaeota was enriched in poplar forest soil. The results of principal coordinates analysis (PCoA) showed that soil bacterial community composition (BCC) of cotton field could be significantly separated from that of poplar forest, and the BCCs in cotton field ecosystem was more consistent. The results of functional prediction showed that nitrification function in cotton field was significantly surpassed the poplar forest. Redundancy analysis showed that the total nitrogen content had a significant effect on the variation of BCC. [Conclusion] Under the influence of long-term cultivation, fertilization and other human activities, the soil bacterial diversity and community composition of oasis cotton field have been significantly changed, the α-diversity of bacterial community was significantly lower than that of the poplar forest, the β-diversity of bacterial community was weakened, and some bacteria related to plant growth, such as Actinobacteria and nitrifying bacteria, have been significantly enriched. This result can provide useful information to utilize and develop Xinjiang oasis ecosystem filed in near future.

Abstract:[Background] Plant-microbial remediation becomes prevailing in the remediation of soil heavy metal pollution. The key to achieve this is to obtain microorganisms which can interact with hyperaccumulator effectively. Solanum nigrum L. is widely used in the remediation of cadmium pollution of farmland. [Objective] To screen cadmium tolerant plant growth promoting rhizobacteria (PGPR) which can promote the growth and cadmium accumulation of Solanum nigrum L. [Methods] Cadmium tolerant strains with good growth promoting characteristics were isolated and screened from rhizosphere soil of Solanum nigrum L. The effects of cadmium stress on plant growth and cadmium accumulation ability were investigated under hydroponic culture, and microorganism which can promote growth and cadmium accumulation of Solanum nigrum L. was determined. The strain was identified by its physiological and biochemical characteristics and the analysis of its 16S rRNA gene sequence. [Results] Four PGPR strains, NT1, AXY1, AW2 and AW1, were isolated from Solanum nigrum L., which were identified as Lysinibacillus sp., Beijerinckia fluminensis, Achromobacter animicus and Herbaspirillum huttiense. The growth of Solanum nigrum L., was enhanced by four strains, as the increase of height, dry matter accumulation and the cadmium accumulation of the aboveground part. Strain NT1 increased height by 31.33%, and increased the dry weight of the aboveground part by 62.65%. Strain AW2 increased the cadmium accumulation by 37.29%. [Conclusion] Screening of strains can provide a practical basis when it comes to improving the efficiency of plant remediation. It can also help to prepare the ecologically functional bacteria and be used for the microorganism and Solanum nigrum L. combination in situ remediation in the cadmium polluted farmland.

Abstract:[Background] In July to August 2018, anthracnose of lettuce occurred in a large area of Wushengyi town, Yongdeng county, Lanzhou city, Gansu province, China. About 40% fields suffered from the disease, and 10% fields failed to harvest. [Objective] Identify the pathogen of anthracnose on lettuce. [Methods] Pathogen isolation was carried out by means of diseased tissue isolation method. Koch’s rule was used to prove the pathogenicity of fungal strain isolated from lettuce. Morphological and molecular biological methods were used to identify the pathogen. [Results] Three fungal strains with similar morphological characteristics, were isolated from diseased samples. Conidia fusiform, with one septum, slightly curved, (10.44?19.40) μm×(2.61?4.48) μm, when the pathogen was cultured on PDA medium for 7 d at 20 °C. Artificial inoculation with representative strain Lett-11 on detached lettuce leaves, induced symptoms similar to in field. BLASTn analysis showed that the ITS sequences of strain Lett-11 (GenBank accession No. MK252097) had a 99% homology with Marssonina panattoniana strain CBS 163.25 (GenBank accession No. MH854831.1). [Conclusion] Pathogen causing anthracnose on lettuce was identified as Marssonina panattoniana [Synonymy: Microdochium panattonianum]. This is the first report of anthracnose of lettuce caused by Mar. panattoniana in Gansu province, China.

Abstract:[Background] Citrulline and ethyl carbamate are hazards generated during rice wine fermentation. From rice wine fermentation broth Lactobacillus brevis 2-34 is isolated with the potential to reabsorb citrulline. [Objective] To identify genes encoding citrulline transporters in this strain will help its effective application in rice wine fermentation. [Methods] With vectors pRSFDuet-1 and pETDuet-1, citrulline metabolism-related enzymes of L. brevis 2-34, including arginine/ornithine antiporter, arginine deaminase, ornithine carbamyltransferase, as well as the two putative citrulline transporters, C4-dicarboxylate anaerobic carrier and AO antiporter, were expressed in Escherichia coli C43(DE3). [Results] Recombinant E. coli had similar citrulline reabsorption ability as L. brevis 2-34. Both putative transporters could absorb extracellular citrulline, and the transporting capability of DcuC was much stronger. [Conclusion] DcuC and AO antiporter are citrulline transporters in L. brevis 2-34.

Abstract:[Background] Lactic acid bacteria, which are widely used in many industries such as food and feed, have become research hotspots for making biological preservatives. [Objective] The antibacterial substance of Lactobacillus plantarum DY6, with the excellent antibacterial effect, was preliminarily explored, which would provide a reference for further application. [Methods] The physicochemical properties of the antibacterial substances in the fermentation broth of Lactobacillus plantarum were studied. The GC-MS metabolomics method was used to analyze the metabolites in the fermentation supernatant, while the main inhibitory substances were speculated by multivariate statistical analysis and initially separated by semi-preparation HPLC for further identification by GC-MS. [Results] Lactobacillus plantarum DY6 had strong inhibitory effect on Staphylococcus aureus, Escherichia coli and Salmonella. The antibacterial ability of fermentation supernatant with different fermentation time was tested. The supernatant of 0?4 h had no antibacterial activity, and the antibacterial ability was gradually increased after 8 h. The antibacterial activity of fermentation supernatant tended to be stable from 24 h to 48 h and was the best at 48 h. The antibacterial diameter was 15.28 mm. The differential markers of lactic acid bacteria fermentation broth were analyzed by multivariate statistical analysis. The main differences were found to be organic acids (such as lactic acid, acetic acid, propionic acid, etc.) and fatty acids (such as caprylic acid and citric acid). Antibacterial components of fermentation supernatant were obtained via semi-preparation HPLC, mainly including: organic acids (such as lactic acid, acetic acid, 3-phenyl lactic acid, phenylpropionic acid, etc.), fatty acids (such as citric acid, octanoic acid, citric acid, etc.), in addition to a small amount of aldehydes and alcohols. [Conclusion] It was determined that the antibacterial substances of Lactobacillus plantarum DY6 were mainly organic acids and fatty acids, which provided a theoretical basis for further antiseptic applications.

Abstract:[Background] Aspergillus oryzae can degrade proteins into nutrients such as peptones, peptides and various amino acids by the action of proteases. An important indicator of broad bean paste, amino nitrogen content is directly affected by protease. Therefore, Aspergillus oryzae plays a key role in the fermentation of broad bean paste. However, we still do not have an appropriative strain for the fermentation of broad bean paste. Thus, the study about high-yield protease fungus special for broad bean paste is meaningful for the development of the entire industry. [Objective] To screen appropriative fungi for broad bean paste and study the effect of fermented broad bean paste compared with Aspergillus oryzae 3.042. [Methods] We screened high yield protease fungi from koji produced by spontaneous fermentation. Then we use these screened fungi as starter to ferment broad bean paste. Then we detected the growth rates of the fungi and the concentration of protease through the koji process. In addition, we also detected the broad bean paste’s amino acid nitrogen and volatile flavor substances in order to analyze the flavor enhancement. [Results] Seventeen strains of high-yield protease were screened. Strain PCSM002 had the fastest growth rate, strain PCSM001 and PCSM002 showed the strongest protease production ability. Protease reached 1 450.25 and 1 703.25 U/g respectively after cultivated 72 h in bran medium. Amino nitrogen content of the fermentation broth with PCSM001 and PCSM002 reached 0.86 μg/kg after 24 days’ fermentation. Broad bean paste fermented by the two strains had rich aroma and perfection in taste. Both strains had been preserved in the Guangdong Provincial Collection of Microbial Strain, and the preserved numbers are GDMCC 60198 and GDMCC 60199 respectively. [Conclusion] The two high-producing protease molds screened in this experiment are expected to be used as the starter of broad bean paste, and provide special fermentation strain for the broad bean paste industry.

Abstract:[Background] Cronobacter spp. are major foodborne pathogens that need to be intensively monitored for food safety. At present, with continuous development of the molecular detection technology, it is very important to develop simple and efficient detection methods for Cronobacter detection in food. [Objective] Develop duplex PCR detection kit for detecting Cronobacter and evaluate its detecting efficacy in food samples. [Methods] Duplex PCR reaction system was optimized and the components of the kit were confirmed. The main reaction reagents were made into powder pattern by freeze drying. Then the specificity, sensitivity, repeatability, shelf life and other performance indexes of the kit were evaluated. [Results] All Cronobacter standard strains and isolated strains were detected positive with two distinct bands in the target size, while all non-Cronobacter standard strains and isolated strains were detected negative without target band observed. The detection sensitivity of Cronobacter purified genomic DNA and pure cultures was respectively 2.3×10?1 ng/μL and 3.2×104 CFU/mL. Cronobacter in 65 food samples was detected. The detection result obtained by the kit was highly consistent with that obtained by conventional microbial detection method. The intra-batch and inter-batch testing repetition rates were all 100%. The kit had no efficacy loss after 120 h storage at 42 °C and 12 months at 4 °C. [Conclusion] The kit is stable, reliable, and applicable for rapid detection of Cronobacter in food.

Abstract:[Background] Lipase is a special ester bond hydrolase and widely used in industries. Microorganisms are the main source of lipase. Many microorganisms in the rumen are of great diversity. Although it has been reported about the production of cellulase by rumen microorganisms, there is no report about lipase produced by rumen microorganisms. [Objective] The purpose of this study was to isolate and screen lipase produced by microorganisms from the rumen of yak, and to identify the strains and characterize the enzymes. [Methods] Olive oil was used as the sole carbon source in neutral red fat medium for preliminary screening, then the obtained strains were rescreened by the improved copper soap-photometric. Finally, the rescreened strains were identified by morphological observation, physiological and biochemical experiments and 16S rRNA gene sequence analysis. The lipases produced by the isolated microorganisms were tested by temperature, pH, metal ions, organic solvents and surfactant, accordingly we had found the optimum conditions. [Results] Six strains with high enzyme activity were obtained. Three were Serratia liquefaciens, two were Geotrichum candidum, and one was Mucor circinelloides. Enzymatic properties showed that the optimal reaction temperature of three lipases (Serratia liquefaciens, Geotrichum candidum and Mucor circinelloides lipase) was 45, 35 and 40 °C, and the optimal reaction pH of them was 8.0, 7.0 and 7.0 respectively. The activities of these lipases were stimulated by Ca2+ and Mg2+ ions and inhibited by Zn2+ ions. Lipases were inactivated by EDTA and SDS. These lipases tolerated glycerol, while the lipase of Mucor circinelloides showed a better tolerance to methanol, ethanol and acetone than others. [Conclusion] We isolated three lipases produced by microorganisms from the rumen of yak. This study showed that rumen microorganisms are of high value in lipase research.

Abstract:[Background] Streptomyces strains are widely distributed in soil environment. With a complex morphological differentiation and a large diversity of secondary metabolic networks, Streptomyces can produce many bioactive secondary metabolites. [Objective] Fatty acid biosynthesis and secondary metabolism are closely related in the model strain Streptomyces coelicolor, but the fatty acid synthetic mechanism is still unclear, and its long-chain 3-ketoacyl ACP synthase has not been reported. [Methods] Through sequence alignment with Escherichia coli FabF (EcFabF), SCO2390 (ScoFabF1), SCO1266 (ScoFabF2), SCO0548 (ScoFabF3) and SCO5886 (ScoRedR) were found in the genome of Streptomyces coelicolor A3(2), which showed high similarity with EcFabF, and contained the conserved Cys-His-His sites, indicating that they may have the 3-ketoacyl-ACP synthase activity. The four genes were amplified by PCR, and ligated into the expression vector pBAD24M, and transferred into E. coli fabB(ts) and E. coli fabB(ts)fabF mutants. The growth of transformants was analyzed. The four genes were also ligated into pET-28b, and expressed in E. coli BL21(DE3). The four EcFabF homologues with hexahistidine-tag were purified by Ni-NTA, and the activities were analyzed in vitro. The fatty acid profiles of E. coli fabF mutants completed with the four genes were also analyzed by GC-MS. [Results] Only ScofabF1 conferred the E. coli fabB(ts)fabF mutant to grow with oleatic acid supplemented at 42 °C, and all failed to complete E. coli fabB(ts) at 42 °C. In vitro enzymatic analysis also demonstrated that only ScoFabF1 has 3-ketoacyl-ACP synthase activity, while the other three proteins showed no similar activity. E. coli fabF mutant harboring ScofabF1 increased the amount of unsaturated fatty acid C18:1 significantly. [Conclusion] All of above suggested that Streptomyces coelicolor ScofabF1 encodes 3-ketoacyl-ACP synthase II, and plays an important role in fatty acid synthesis. However, no gene encoding 3-ketoacyl-ACP synthase I was found in the genome, indicating that Streptomyces coelicolor may have other mechanism to synthesize small amount of unsaturated fatty acids. Achievement in this study will contribute to further research about the mechanism of fatty acid synthesis in Streptomyces coelicolor.

Abstract:[Background] Natamycin is a natural, broad-spectrum and efficient polyene macrolide antifungal antibiotic. Streptomyces gilvosporeus is an important natamycin-producing bacterium. However, the genome sequence analysis of S. gilvosporeus has not been reported until now, limiting studies on the biosynthesis and regulation of natamycin and other secondary metabolites in S. gilvosporeus. [Objective] The genomic sequence information of the S. gilvosporeus F607 (a natamycin high-producing strain) was analyzed to explore the genetic resources of secondary metabolite genes and lay a foundation for further study on the mechanisms of high producing and regulation of natamycin biosynthesis. [Methods] The genome sequence of F607 was analyzed with softwares to predict genes, to annotate function of genes, to analyze phylogenetic tree and colinear analysis, and to predict the secondary metabolite synthetic gene cluster; The differences of natamycin biosynthetic gene clusters in different strains were analyzed and compared through annotating analysis of natamycin biosynthetic gene clusters; the biosynthetic pathway of natamycin was analyzed and predicted according to gene function. [Results] The complete genome sequence of F607 is 8 482 298 bp ((G+C)mol%, 70.95%). 5 062, 4 428, 5 063 genes were respectively predicted in COG, GO and KEGG databases. The antiSMASH software predicted that there are 29 secondary metabolite biosynthetic gene clusters in F607 genome. The homology of natamycin biosynthetic gene cluster in F607 shared 81% and 77% with that in S. natalensis and S. chattanoogensis respectively. Although there were differences among 2 regulatory genes (sngT, sgnH) and 9 unknown function genes (orf1?9), the analysis of natamycin biosynthetic gene clusters in different strains indicated that the other genes and their arrangement in natamycin biosynthetic gene cluster are highly conserved. [Conclusion] In this study, the complete genome sequence of strain F607 was first analyzed and the biosynthetic pathway of natamycin of F607 was predicted. This study provides basic data for analysis of the molecular mechanism of high-yield natamycin in S. gilvosporeus F607, and aids in the development of useful strategies for revealing the mechanism of high yield of natamycin, improving industrial strains and innovating drug discovery.

Abstract:[Background] Microbial transformation is an important means for structural modification of natural products, featured with fast reaction, high selectivity, easy-controlled conditions and little pollution. Many microorganisms have been isolated for transformation of bufadienolides and several derivatives of reduced toxicity can be obtained by the modification at different positions. [Objective] The aim of this work is to find compounds with better bioactivity or reduced toxicity by screening the converting strains towards bufalin. [Methods] Substrate transformation experiments were carried out with bufalin as substrate to screen strains, and the products were identified by HPLC (High performance liquid chromatography) and LC-MS (Liquid chromatograph-mass spectrometer). The selected strain was determined in terms of colony morphology observation and molecular biological identification. The fermentation conditions were optimized to increase the conversion rate. The transformation of other steroids were also determined. [Results] One bufalin-transforming strain was obtained and identified as Naganishia sp. by the morphological characterisitics and evolutionary analysis on the basis of ITS sequence. The transformation product of this strain to bufalin was 3-ketobufalin. The optimal conversion conditions were determined with the initial pH value of 6.5, substrate concentration of 8 mg/L, the inoculation amount of 3% and the transformation time of 96 hours. About 48.3% of bufalin was converted into 3-ketobufalin. In addition, this strain could also transform the reversible reaction of estrone and 17β-estradiol. [Conclusion] It is first reported that Naganishia strain can transform bufalin into 3-ketobufalin, with less toxicity and great potential for safe drug to heart failure. The capability of transforming steroids of this strain also makes it possible to bioconvert and modify other steroidic compounds. The microbial conversion can provide a convenient path for large-scale production of the valuable compound because of high selectivity, mild reaction conditions and simple operation process.

Abstract:[Background] In recent years, with the continuous development of manned spaceflight, the issue of space microbial safety cannot be ignored. The disease spectrum of manned spaceflight indicates that infectious diseases have great impact on the health of astronauts. Escherichia coli is a normal flora in the human intestinal tract. Meanwhile, it can cause intestinal infection under certain conditions and become a conditional pathogen. It is of great significance to analyze the effect of space environment on the metabolism of E. coli. [Objective] E. coli was carried on the “SJ-10” satellite for 12 days to analyze and identify the changes in the metabolic level of E. coli in the space environment. [Methods] The non-targeted metabolomics technique was used to collect data and perform mass spectrometry analysis from E. coli carried on the satellite. At the same time, a ground control group was set up. Principal component analysis and orthogonal partial least squares discriminant analysis were used to identify potential differential biomarkers between groups. [Results] A total of 12 significant biomarkers were found by analysis and identification. In the spaceflight group, 11 biomarkers involved in energy metabolism and glycerophospholipid metabolism were up-regulated, and one fatty acid content was down-regulated. [Conclusion] Space flight can improve the level of energy metabolism and glycerophospholipid metabolism of E. coli, and the changes of related metabolites suggest that space flight may promote the proliferation of E. coli.

Abstract:[Background] Lactobacillus has been proved to adsorb many cancerogens, but there are little studies on the mechanism of the adsorption of Lactobacillus to benzo(a)pyrene (BaP). [Objective] The adsorption capacity and mechanism of Lactobacillus pentosus ML32 and Lactobacillus plantarum 121 on BaP in processed meat products were investigated. [Methods] The adsorption rate of BaP from different processed meats was detected by HPLC. [Results] BaP adsorption rates in meats treated by frying, smoking or grilling, of both Lactobacillus plantarum 121 and Lactobacillus pentosus ML32 were more than 30%. The adsorption rate of strain 121 in the directly smoked meat was 41.21%, and that of directly fried meat was 38.71%. The adsorption rate of strain ML32 to BaP in the indirectly smoked meat was 40.02%, and that of indirectly barbecued meat was 38.01%. In conclusion, L. plantarum 121 shows an ability to remove more BaP in the meats processed with high temperatures or a long time, while L. pentosus ML32 seems to be beneficial in reducing BaP level in the low-temperature or short-time treatments. In addition, peptidoglycan in the Lactobacillus cell wall may play an important role in the adsorption process. [Conclusion] L. plantarum 121 and L. pentosus ML32 have the effect of removing BaP from processed meat products and can be used as a method to reduce the risk of excessive BaP in meat products.

Abstract:[Background] The preservation method and its effect are the premise and guarantee for the quality of strains. It is more and more urgent to select the best preservation method for strains of Hypsizygus marmoreus with the increasing of cultured scale and yield year by year. [Objective] Some common preservation methods and their effects were analyzed and studied in order to find a simple, efficient and inexpensive preservation method for strains of Hypsizygus marmoreus. [Methods] The preservation effect of each preservation method was evaluated by comparing the growth rate of mycelial, dehydrogenase activity of mycelial and the decolorization rate of mycelium to LBL medium. [Results] By comparing and analyzing the three-month preservation effects of three kinds, 27 different preservation methods of Hypsizygus marmoreus strains, it was found that the best preservation method was water solution preservation, among which 0.1% PEG6000 water solution was the best; the second was sawdust preservation method, among which the saline-soaked poplar sawdust was the best; The slant preservation method was the third. The effect of normal preservation experiment was better than that of accelerated preservation experiment. [Conclusion] Compared with the conventional preservation methods for strains of Hypsizygus marmoreus, the preservation methods adopted in this experiment greatly broadened the types and scope of the preservation methods, and improved the preservation effects of strains.

Abstract:Cyclic diadenosine monophosphate (c-di-AMP) is a ubiquitous class of second messenger molecule in bacteria, whose metabolism is finely tuned by diadenylate cyclase (DAC) and phosphodiesterase (PDE). c-di-AMP is not only involved in many essential processes such as cell growth, cell wall homeostasis and ion transport, but also in host anti-bacterial immunity through being sensed by eukaryotic sensors/receptors of host cells. In particular, c-di-AMP has been found to play important roles in regulating host innate immunity such as type I interferon response, activation of NF-κB signal pathway, autophagy and inflammatory response. Acting as a mucosal adjuvant, c-di-AMP induces host adaptive immune response as well. Thus c-di-AMP is now considered to be a newly identified pathogen associated molecular pattern (PAMP), which becomes a new target in bacterial vaccines and drug research.

Abstract:Biological wastewater treatment systems are driven by microbial physiological processes, metaomics approaches obtain information from different molecular levels, providing a new way to understand microorganism in wastewater treatment systems. In this review, we summarize the developments of metaomics such as metagenomics, metatranscriptomics, metaproteomics and metabolomics, highlight the current research of metaomics and integrated metaomics in wastewaters treatment systems, also indicate the prospect in practical application.

Abstract:The gastrointestinal flora plays an important role in animal health and production performance. The application of probiotics in livestock and poultry can regulate intestinal micro-ecological balance, regulate fat metabolism, improve feed utilization and promote the formation of flavor substances. It can improve meat quality, included carcass quality of livestock and poultry. This paper reviews the mechanisms what regulates gastrointestinal microecology by different probiotics, and improves the meat quality of livestock and poultry by improving the structure of intestinal flora. It can reference for further research and development of probiotic feeds targeting gastrointestinal flora.

Abstract:As a microbial flora distributed in the human intestine, the intestinal flora plays an important part in the normal physiological function of the intestine. Clinical studies have revealed that extensive use of broad-spectrum antimicrobials breaks the balance of the intestinal flora, leading to antibiotic-associated diarrhea (AAD). However, the relationship between different intestinal flora and AAD is not identical. This paper reviews the mechanism of AAD caused by pathogenic bacteria, the principle of prevention and treatment of AAD by probiotic, and the relationship between conditional pathogens and AAD in order to provide theoretical basis for the study of intestinal flora and AAD. At the same time, it provides references for more accurate prevention, diagnosis and treatment of AAD.

Abstract:Myxobacteria are Gram-staining-negative and rod-shaped bacteria belonging to the order Myxococcales in the class of Deltaproteobacteria. The excellent capability of producing secondary metabolites makes myxobacteria one of the most important sources of natural products, following fungi and actinomycetes. However, the research and development of myxobacteria has been limited because of the difficulty to isolate and purify myxobacteria strains. With the advances in sequencing technology and bioinformatics, a large number of genomes of myxobacteria have been sequenced and released. In this paper, we reviewed the importance of research, value of resource development and difficulties of isolation and purification in myxobacteria, summarized the published myxobacterial genomes and their annotation information, introduced the research progress of myxobacterial genomics involved in myxobacterial ecology, predation mechanism, formation of fruiting bodies and producing of secondary metabolites. This paper is helpful for understanding the important value of genomics in myxobacteria research and for further investigating the metabolic mechanism and social behaviors of myxobacteria using multi-omics techniques, with great significance for resource collection and development of myxobacteria.

Abstract:In recent years, the relationship between gut flora and psychiatric disorders such as autism spectrum disorder, anxiety, depression, has gained increasing attention. This article mainly introduced the correlation between gut flora and depression, and the existing evidence that whether and how the probiotics might affect depression. Although exciting progress has been achieved in this area, further studies are still needed to promote our understanding about the causality between gut flora and depression, and to fully assess the potential of probiotics to prevent and treat depression.

Abstract:Natural products and their derivatives, including the compounds designed based on the structure of natural product pharmacophores, account for more than 50% of approved drugs. The medicinal value of fungal natural products has been known for centuries, and millions of patients worldwide have been treated with fungal-derived small molecule drugs every year. Fungal-derived small molecule drugs are very valuable, both in terms of market prospects and humanitarian perspectives. This review summarizes the clinical application of fungal natural products, and the development of statins reveals that fungal-derived natural products are an important source of inspiration for drug synthesis in the 21st century. This review covers small molecules of fungal-derived drugs, including natural drugs, related derivatives, and structurally modified drugs.

Abstract:Catechol is one of the most important intermediate products in the various pathways in degrading polycyclic aromatic hydrocarbons (PAHs). The ring cleavage can occur in two different orientations relative to the vicinal diols, and this difference in cleavage site is typically used to classify the catechol dioxygenases into two groups, the intradiol- and extradiol-cleaving enzymes. The essential process of intradiol pathway is the intradiol ring-cleavage catalyzed by catechol 1,2-dioxygenases. This paper summarized the research advances in these fields with the focus on the structure, enzymatic property and isoenzymes of the catechol 1,2-dioxygenases.

Abstract:In recent years, as a student-centered and teacher-oriented teaching method, flipped classroom has been gradually applied in undergraduate education. This study focused on the problems existing in the current experimental courses of medical microbiology, introduced the basic method and implementation effect of using a flipped classroom based on microlectures. Compared with the traditional teaching methods, the reformed teaching method could improve the teaching quality significantly, thus providing useful experience for the reform of other basic medical courses in our university.

Abstract:Agricultural microbiology plays an important role in the sustainable development of modern agriculture. In order to keep pace with the profound innovation and promotion of the modern agricultural industry in China nowadays, high-tech specialists will be highly in demand. Therefore, the content of Microbiology course curriculum is renovated and optimized according to the feedback from the collaborating enterprises. An online/offline integrated mode has been adopted, both theoretical and experimental course have been integrated to reinforce students’ understanding and application of agricultural microbiology techniques. The renovated course will be more practical and straightforward to prepare current students for their future application in modern agricultural industry.