Abstract:[Background] Straw mushroom (Volvariella volvacea) is one of the most varieties of edible fungi with Chinese characteristics, and its consumption increased year by year, the potential industrial development for V. volvacea is tremendous. Liquid spawn used in straw mushroom cultivation is the development trend of factory production, the current research in liquid spawn of straw mushroom are mainly focus on the optimized formula and growth condition, however, less research was done on the mycelial activity during the cultivation process. [Objective] Study the physiological changes of straw mushroom in liquid spawn cultivation, and analyze the cultivation end-point. [Methods] Since cultivation 48 h, collection samples every 12 h, the biomass and protein content of mycelia were measured in 168 h. Moreover, pH value, sugar degree, reducing sugar contents, cellulase, hemicellulase and laccase activities of liquid spawn filtrate were determinated and analyzed in the cultivation process. [Results] The results showed that after 84 h of cultivation, the growth rate of mycelia diminishing and the biomass increased slowly, the pH value, sugar degree and reducing sugar contents reduced gradually, cellulase and hemicellulase activities also declined gradually. After 96 h, the protein content of mycelia declined while protein content of filtrate rised obviously. [Conclusion] The research on physiological changes of 9715 liquid spawn indicated that 84?96 h was the cultivation end-point, and the results can provide important reference value for liquid spawn application in the straw mushroom 9715 factory production.

Abstract:[Background] The types and electrochemical activity mechanism of exoelectrogenic microorganisms have important influence on the electrogenic performance of microbial fuel cells. [Objective] A salt-tolerant exoelectrogenic strain was isolated from seawater, its exoelectrogenic characteristic was studied and the species information was identified. [Methods] A pure exoelectrogenic bacterium (designated as E-1) was isolated from the enriched anodic biofilm of microbial fuel cells (MFCs), where the seawater from the south sea in China was used as the inoculum and MFCs were operated in the anolytes without and with salt addition. The exoelectrogenic characteristic of the pure isolate strain E-1 was investigated by inoculating into MFCs containing different salt concentrations. The species information was identified using the combined methods of morphological observation, Biolog analysis and 16S rRNA gene sequence alignment. [Results] The power densities of strain E-1 were respectively 51.69 mW/m2 and 26.56 mW/m2 without salt addition and with addition of 6.6% NaCl, which was related to its good salt tolerance. The strain E-1 was identified as Shewanella algae, which showed diversity in substrate utilization, and its growth temperature and pH range were 25?40 °C and 5.0?10.0, respectively. [Conclusion] The strain affiliated to S. algae is firstly reported to show electricity activity when applying in MFCs, which enriches the diversity of electricigens. S. algae E-1 can generate electricity at high salt concentration, which provides a new experimental material for the application of MFCs in seawater resourceful treatment.

Abstract:[Background] Tobacco aging is a process involving the interaction of many factors. [Objective] In order to investigate the relationship between the structure, function and chemical composition of culturable microflora on the surface of aging tobacco leaves. [Methods] The microflora of tobacco leaves stored in Guiyang, Tanchang and Maotai storerooms were isolated at different aging time respectively. The function of bacteria and fungi were analyzed by annotating to the FAPROTAX and FUNGuild databases respectively. At the same time, combining with the main chemical components of tobacco samples, the correlation analysis of culturable microflora was carried out. [Results] The results showed that 189 strains of dominant bacteria and 229 strains of dominant fungi were isolated from 243 tobacco leaf samples. Altogether, Bacillus was the dominant population in bacteria, whereas Aspergillus and Penicillium were accounted for dominant fungal populations. With the extension of aging time, the proportion of dominant populations and dominant functional groups were decreased gradually, and in the other side, the main chemical substance was correlated with the change of microbial communities significantly. [Conclusion] It is demonstrate that the structural changes of microbial functional groups have a contribution to accelerate tobacco aging process. In turn, the changes of major chemical components during the aging process could affect the composition and function of microbial communities.

Abstract:[Background] The unique environment of the ocean creates a diversity of marine life, and bacteria in marine sediments play a vital role in the marine environment. [Objective] To study the similarities and differences of bacterial communities between terrestrial soil and marine sediments, so as to better understand the diversity of marine bacteria and the potential role of sediment bacteria in the marine environment. [Methods] Marine sediment samples and terrestrial soil samples were collected from the Yellow Sea waters of China and the Dahei Mountain in Dalian by 16S rRNA gene high-throughput sequencing technology. The bacterial community structure of marine sediments was analyzed by using terrestrial soil as a control. [Results] The abundance of Bacilli, Sphingomonas and Bacillus in marine sediment samples was higher than that in terrestrial soil samples. The abundance of uncultured bacterium f. Nitrosomonadaceae, and uncultured bacterium f. Anaerolineaceae in marine sediments is lower than that in terrestrial soil, but the abundance values are also higher than 1%. Sample taxonomic statistics show that the sequence abundance ratio of Acidobacteria in marine sediments and terrestrial soil samples is large, and the sequence abundance of Sphingomonas in marine sediment samples is greater than that of terrestrial soil samples. [Conclusion] Marine sediment bacteria diversity can be used as an important index of marine environment recovery, the study provides theoretical basis for rational development and utilization of marine resources.

Abstract:[Background] Microorganisms are frequently subjected to multiple stresses in deserts, including drought, high temperatures and UV radiation. These natural stressors make it easy for desert soil microorganisms to accumulate numerous superoxide ions and peroxides inside and outside their bodies, inhibiting their growth or directly causing death. [Objective] Desert soil bacteria exhibit antioxidant properties in order to adapt to the desert environment. As an important part of the desert ecosystem, the research on its antioxidant properties provides the scientific and technical bases for the development of antioxidant resources in desert areas, as well as the antioxidant mechanism of desert microbes. [Methods] We used desert soil bacteria as the research object to obtain two bacteria with strong antioxidant activity by hydrogen peroxide: Planomicrobium okeanokoites AX6 and Kocuria marina KD4. The antioxidant physiological and biochemical characteristics of bacteria in desert soil was explored by its growth curve under hydrogen peroxide, cell damage, antioxidant enzyme activities and free radical scavenging ability. [Results] The content of malondialdehyde in the cells of the two strains of bacteria in low concentration of hydrogen peroxide was significantly lower than that in negative control Escherichia coli. The glutathione peroxidase activity of strain AX6 was higher at 108.33 U/mL in 1.5 mmol/L hydrogen peroxide, meanwhile the scavenging ability of DPPH and superoxide anion radical was higher; while the catalase activity of strain KD4 increased to 1.16 U/mL in 3 mmol/L hydrogen peroxide was significantly higher than the positive control Deinococcus radiodurans, as well as the ability to scavenge hydroxyl radicals was higher. [Conclusion] There are large differences in the active antioxidant enzyme and free radical scavenging ability of bacteria in different desert soils, indicating the diversity of microbial antioxidant processes in desert soils.

Abstract:[Background] The accumulation of plastic wastes is an increasingly serious environmental pollution problem, and PVC, as common plastic, has a large number of production and consumption. [Objective] Based on the phenomenon that Tenebrio molitor feeds on plastics naturally, in this paper, the biodegradation of polyvinyl chloride (PVC) plastics by Tenebrio molitor larvae and its intestinal microbes was researched. [Methods] The weight increase of Tenebrio molitor larvae was observed after feeded polyvinyl chloride only, and fourier transform infrared spectroscopy (FTIR), screening of intestinal microbes and high-throughput sequencing methods were used to research biodegradation of PVC. [Results] The results showed that when PVC was used as the only carbon source of Tenebrio molitor larvae, after 32 d, PVC was consumed at the weight of 0.499±0.023 g per 200 Tenebrio molitor larvae, and the average weight of 200 Tenebrio molitor larvae was increased by 0.015±0.002 g. FTIR was used to detect the significant increase of carbonyl and hydroxyl content in PVC from excrement components, which indicates PVC degraded obviously. The results of high-throughput sequencing showed that Hafnia, Morganella, Escherichia coli and uncultured Enterobacteria were the dominant microflora. Compared with conventional feeding control, the abundance of Hafnia and Morganella increased by 35.20% and 16.42% respectively. [Conclusion] This article proved the biodegradation of PVC by Tenebrio molitor larvae and its intestinal microbes, and Hafnia and Morganella strains had the highest utilization of PVC. The results of the study have enriched scientific evidence for the biodegradation of “white pollution”.

Abstract:[Background] Eutrophication of plateau lakes is becoming increasingly serious. Lakeshore zone is the protective barrier for the lake and has effects on intercepting and purifying the external pollutants. Changes of water environment affect the sediment bacteria. [Objective] The aim of the present work was to explore the relationship between lakeshore sediment bacterial community structural characteristics and water bodies eutrophication. [Methods] Bacterial community structure and the diversity of eight sediments in the south bank of Yangzonghai Lake was analyzed by 16S rRNA gene high-throughput sequencing technology. Principal component analysis and redundant analysis were analyzed by comparing the differences in environmental factors of different waters, and the effects of eutrophic water on bacterial community structure and abundance in sediments. [Results] Lakeshore sediments had a certain response to water eutrophication. In high eutrophication areas (S3), the bacterial richness was high with a high OTU at 1 473. Conversely, in areas with low levels of eutrophication (S1), the bacterial richness was low and the OTU was 730. The dominant bacteria in Yangzonghai Lake lakeshore were Proteobacteria and Chloroflexi with a small group of Actinobacteria, Actidobacteria and Firmicutes. Chloroflexi was closely related to eutrophication degree in water. In moderately eutrophic areas, the proportion of Chloroflexi was as high as 44.1%, while in the light eutrophication area Chloroflexi was only 15.6%. Basing on environmental factor analysis, it was found that the bacterial community of Yangzonghai Lakeshore sediment was strongly affected by total phosphorus, chlorophyll a and total nitrogen. [Conclusion] The results clarified the structure and variation characteristics of bacterial populations in plateau lakes lakeshore and their responses to water eutrophication, helps to better the understanding of sediment bacteria in plateau lakes and provide a theoretical basis for prevention and control eutrophication in plateau lakes.

Abstract:[Background] High-throughput sequencing methods are widely used in analyses of environmental microbial communities and diversities, and make it easy to investigate endophytic fungi in plants. However, choices of primers often lead to difference in results because fungal species detected depend on fungal coverage rate of primers. Plants in the genus Salicornia are one kind of the most salt-tolerant plants. Some endophytic fungi isolated from the plants produced important functional metabolites. But few investigations on the diversity of endophytic fungi were reported. [Objective] To investigate the endophytic fungi community in Salicornia europaea and reveal effects of different PCR primers on high-throughput sequencing analysis. [Methods] Plants of Salicornia europaea were sampled from Guhya Salt Lake of Urumqi county in Xinjiang, and ITS DNA of endophytic fungi was amplified with primer pairs of ITS1-5F, ITS1-1F and ITS2. Then OTUs based on high-throughput sequencing were analyzed. [Results] In total 102 OTUs were obtained involving 8 phyla and unclassified flora in fungal domain. Among them, Ascomycota was dominant phylum followed by Basidiomycetes. A total of 64 genera and 20 unclassified genera were discovered, in which Alternaria, Cladosporium and Podospora were major groups. Effects of different PCR primers on high-throughput sequencing were measured. In total of the 102 OTUs, 44 OTUs were obtained from the ITS1-5F primer pair, 55 OTUs from the ITS1-1F primer pair and 25 OTUs from the ITS2 primer pair, whereas only 5 OTUs were detected at the same time by all three primers pairs. Additionally, ITS1-1F primer pair was recommended with ITS1-5F primer pair as the supplementary strategy in investigation of the diversity of endophytic fungi. [Conclusion] There are abundant resources of endophytic fungi in Salicornia europaea and obvious effect on analyzing the composition and distribution of endophytic fungi in high-throughput sequencing.

Abstract:[Background] In recent years, the polylactide/polybutylene adipate-co-terephthalate (PLA/PBAT) mulch films has been widely used. However, the effects of PLA/PBAT mulch films on bacterial community structureits has rarely been reported. [Objective] The effects of PLA/PBAT mulch films on bacterial community structure was studied. And we isolated the PLA/PBAT degrading bacteria to provide technical support for in situ remediation. [Methods] The high-throughput sequencing method was used to compare the structural changes of bacterial communities in the Xinjiang soil before and after using the PLA/PBAT mulch films. The PLA/PBAT degrading bacteria were isolated by screening medium, and the degradation effects of the strains were studied by different culture conditions. [Results] After using PLA/PBAT mulch films, the abundance of Acidophilus and Bacillus were increased, and the abundance of Proteobacteria and Actinomycetes were decreased. This might be due to the inhibitory effect of intermediate products on pH and microorganisms during the degradation process of the PLA/PBAT mulch films. Moreover, a degrading strain XJ11 was isolated from the soil, which was initially identified as Delftia tsuruhatensis. In the screening medium, 1.5% tryptone was added, 1 mL of seed solution was inoculated, the pH of the medium was adjusted to 7.2, and shaker conditions was adjusted to 37 °C and 130 r/min. The degradation rate of PLA/PBAT mulch films (specification: 1 cm×1 cm×0.05 cm) can reach 6.87%. [Conclusion] The PLA/PBAT mulch films could change the bacterial community structure of soil. Screening the PLA/PBAT degrading bacteria from the environment was an effective measure to solve the pollution of mulch films.

Abstract:[Background] The use of traditional flocculants poses security and environmental problem, while microbial flocculants have the advantages of non-toxic, no secondary pollution and easy biodegradation. Therefore, it is of great significance to find efficient and economical microbial flocculants. [Objective] To study the flocculation characteristics and mechanism of PCF-1767, an extracellular polysaccharide flocculant produced by Phanerochaete chrysosporium BKMF-1767. [Methods] The flocculating activity of the PCF-1767 was determined using a Kaolin suspension. The effects of flocculant acquisition time, flocculant dosage, pH, Ca2+ concentration and temperature on the flocculation efficiency were studied, as well as the thermal stability of PCF-1767. Flocculation mechanism of PCF-1767 was discussed from the monosaccharide composition and glycoside bond connection type, the molecular weight of polysaccharides, Zeta potential determination, and flocs morphology observation. [Results] The PCF-1767 obtained at 6–12 d displayed high flocculating activity. For higher flocculation activity of the PCF-1767 obtained at 7 d, at least 2 mmol/L Ca2+ should be added as coagulant. The optimal flocculant dosage was 0.75–1.35 mg/L, with an optimum pH range of 3.0–9.0. The flocculating activity decreased when the water temperature was higher than 50 °C. The flocculation activity of PCF-1767 was almost unchanged after 3 h water bath treatment. The primary structure of the polysaccharide in PCF-1767 obtained in different periods was similar, mainly connected by glucose in the manner of →4)Glup(1→. The molecular weight of PCF-1767 increased with the culture time, at the range of 9.897×105–2.126×106 Da. Zeta potential determination and flocs image analysis showed that the main flocculation mechanism was adsorption bridging flocculation and sweeping, instead of charge neutralization. [Conclusion] The flocculant PCF-1767 produced by P. chrysosporium BKMF-1767 has high flocculation efficiency with low dosage, wide application range and good thermal stability. In brief, PCF-1767 has good application prospects.

Abstract:[Background] 16S rRNA gene sequencing is an important method to investigate microbial communities inhabiting diverse environments. [Objective] This study aimed to reveal the influences of 16S rRNA gene V4 and V3?V4 sequencing with sequencing depth of 10 000?20 000 and 100 000 on analyzing bacterial communities inhabiting oil reservoirs. [Methods] The number of the bacterial 16S rRNA gene copies of the sample used here was (6.51±0.56)×108/L. The bacterial 16S rRNA gene amplicons were sequenced on MiSeq PE250 and PE300 sequencing platforms, respectively. [Results] The Good’s coverage of each sequencing library reached up to 99.6%, when the sequencing depth reached 10 000. The data obtained by V4 and V3?V4 sequencing were repeatable. The α diversity indices of the communities obtained by V4 sequencing with depth of 10 000?20 000 and 100 000 did not show significantly differences. Furthermore, there were few microbial populations that showed significant differences. Compared with the communities obtained by V4 sequencing, lower α diversity indices were observed in the communities obtained by V3?V4 sequencing with a same sequencing depth, and the relative abundances of Epsilonproteobacteria (51.37%:64.23%) and Deltaproteobacteria (17.96%:11.40%) had significant differences. [Conclusion] The results indicated that increasing sequencing depth had a more distinct impact on community α diversity analysis than community β diversity analysis for a given sample, differential α diversity indices and some dominant microbial populations with significantly differential relative abundances were observed between the communities obtained by V4 and V3?V4 sequencing. In view of the increase of sequencing length and lower cost of MiSeq sequencing, higher sequencing coverage and more 16S rRNA bases, V3?V4 sequencing is suggested as the preferred method at the current stage.

Abstract:[Background] Carbonic anhydrase has become the hotspot in carbon reduction research due to its ability to efficiently convert CO2 to HCO3－. Because of the flue gas high temperature, searching for thermostable, the thermophilic carbonic anhydrase is the key to achieve the biomimetic capture of CO2 in industrial flue gas. [Objective] The carbonic anhydrase (CAH) gene of Thermosynechococcus elongatus PKUAC-SCTE542 (Ecah) and Synechococcus lividus PCC6715 (Pcah) were cloned and expressed in Escherichia coli, and the enzymatic properties were characterized. [Methods] The two genes of the CAH were amplified by PCR. The recombinant plasmid pETM11-ECAH, pETM11-PCAH was overexpressed in BL21(DE3) pLysS by IPTG induced. The recombinant carbonic anhydrase was purified with Ni-Agarose His-tagged affinity chromatography and the enzymatic properties were further checked. [Results] Two length of 534 bp carbonic anhydrase were both obtained from E542 and PCC6715. The CO2 hydration activity of the ECAH and the PCAH was 42.6 WAU/mg-protein, 47.6 WAU/mg-protein, respectively. After 50 °C incubation for 30 min, the ECAH activity was increased by 8%, but the PCAH was decreased by 10%. The ECAH activity was increased to about 108% after treated by Ca2+ for 30 min, but no significant inhibition of PCAH activity was observed. Both the ECAH and PCAH activities were significantly inhibited by Zn2+ and sulphanilamide. [Conclusion] The ECAH of the Thermosynechococcus elongatus PKUAC-SCTE542 showes favorable thermostability than PCAH of the Synechococcus lividus PCC6715. EACH meets the basic requirements for CO2 treatment of industrial high-temperature point source flue gas, and enriches the thermophilic carbonic anhydrase gene pool.

Abstract:[Background] Fruit rot caused by Penicillium choerospondiatis is the main postharvest disease of Phyllanthus emblica L., and frequently causes serious fruit decay and large economic loss. [Objective] To obtain and evaluate the control effects of actinomycetes strains with strong antagonistic effect on P. emblica rot fungi. [Methods] Agar disk method and growth rate method were used to screen antagonistic strains against P. choerospondiatis DQ23, and the morphological, physiological and biochemical characteristics combined with 16S rRNA gene sequence were used for classification and identification. The biocontrol effect of the cell-free fermentation broth on fruit rot and quality of P. emblica was evaluated. [Results] Strain SC-15 had a strong antagonistic effect on P. choerospondiatis DQ23 mycelial growth with inhibition zone diameter of 18.70 mm. The inhibition rate of cell-free fermentation broth on mycelial growth of P. choerospondiatis DQ23 reached 87.8%. Based on the morphological and physiological characteristics and 16S rRNA gene sequence analysis, strain SC-15 was identified as Streptomyces virginae. The disease percentage and disease severity index of P. emblica fruit decreased significantly after the treatment via soaking in cell-free fermentation broth of strain SC-15 before storage. The decrease rate of soluble solids and titratable acids in the treated fruits was also slowed down. The results showed that 75% of cell-free fermentation broth had the best protection against P. emblica fruit rot. On the 8th day of storage, the fruit disease percentage was 53.3%, the fruit disease severity index was 35.2%, the content of soluble solids and titratable acid were 4.8% and 6.25 mg/g, respectively. [Conclusion] Streptomyces virginae SC-15 can effectively prevent postharvest fruit rot of Phyllanthus emblica caused by P. choerospondiatis.

Abstract:[Background] Pestalotiopsis trachicarpicola is a new pathogen causing black spot disease of Eucommia ulmoides. There is no report that biocontrol bacteria had antagonistic activity on it. [Objective] The best Bacillus sp. antagonistic activity against P. trachicarpicola DZHBB-1 was isolated and selected from healthy leaves of E. ulmoides, the rapid and accurate identification of this strain was achieved by using the 16S rRNA gene binding protein-encoding gene. [Methods] The strains were isolated from healthy E. ulmoides leaves by dilution separation method, screened by sputum experiments and growth rate methods, combined with 16S rRNA, gyrA and gyrB gene sequences to construct phylogenetic tree analysis. The target strains were identified by its morphological characteristics, physiological and biochemical properties. The pot experiment was carried out to verify its control effect on black spot of E. ulmoides. [Results] A total of 62 strains of Bacillus were isolated and 14 strains with good biocontrol effect on pathogens were screened out. The results of rescreening showed that the strain J1 had the best antagonistic activity and was stable, the inhibition rate reached 66.67%. Phylogenetic tree analysis of 16S rRNA, gyrA and gyrB gene sequences showed that strain J1 was B. amyloliquefaciens. In the pot experiment, the strain could effectively prevent the occurrence of black spot disease of E. ulmoides and the control effect exceeded 57%. [Conclusion] This strain has the potential to prevent and cure the related plant diseases caused by P. trachicarpicola. 16S rRNA, gyrA and gyrB genes are combined to identify Bacillus sp., which can provide a reference method for the rapid and accurate identification of Bacillus sp. and its related species.

Abstract:[Background] Citrus is an important economic crop in rural areas of South China. Due to the influence of the postharvest fungi, a large number of fruits are rotted and deterioratedannually resulting in huge economic losses and seriously affecting the enthusiasm of citrus farmers. [Objective] The filamentous fungus SG-4 exhibited strong antagonism to the postharvest decay mold Penicillium citrinum and Penicillium digitatum. In order to figure out the fungicidal substance, here we performed biological identification and active ingredient analysis. [Methods] The internal transcribed spacer (ITS) of strain SG-4 nuclear ribosomal DNA was sequenced to identify the strain together with the morphology. The antifungal activities of the mycelium phase and culture liquid were traced and the main active substance was separated by alcohol precipitation and reverse high performance liquid chromatography (RP-HPLC). The structure was determined by high resolution mass spectrometry and nuclear magnetic resonance spectroscopy. The antimicrobial activity was evaluated both by Oxford cup method as in vitro and by inoculation test of citrus as in vivo. The content of the fungicidal substance in fermentation broth was determined by RP-HPLC. [Results] Molecular and morphological identification showed that the strain SG-4 was a Penicillium oxalicum. The active substance inhibiting postharvest decay mold of citrus in the aqueous extract was highly polar. Further studies showed that the substance was a novel linear alcohol-soluble pentapeptide, which contained three hydroxyl amino acids and was rich in methylation modification. The first amino acid was beta-aminobutyric acid (BABA), and the linear structure was O-Me-BABA-N-Me-Thr-N-Me-Val-Ser. The inhibitory effect to postharvest decay mold is significantly better than the positive control, and the initial content in the culture liquid got to 90 mg/L, reached the level of industrial production. [Conclusion] The new linear pentapeptide from P. oxalicum showed strong antagonism to citrus decay fungi. Since it is the first time to report both the structure and the activity at home and abroad, the pentapeptide can be developed as an antibiotic to inhibit fungi in agriculture and food industry in the future.

Abstract:[Background] Bacillus velezensis HN-Q-8 has biocontrol effect on Rhizoctonia solani effectively. [Objective] Identify the inhibition substances produced by Bacillus velezensis HN-Q-8. [Methods] Both effects of strain HN-Q-8 on five pathogens of strain potato and stability of fermentation broth were detected though the growth rate method. Active substances of HN-Q-8 were identified using matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS). [Results] The fermentation broth of strain HN-Q-8 exhibited strong inhibitory activity against Rhizoctonia solani, Fusarium oxysporum, Alternari solani and Phytophthora infestans, with the inhibition rates ranging from 50% to 90%. The inhibition rates of fermentation broth retained 74% after 35 min exposure to UV, and 92% after a 10 hour exposure to natural light, respectively. The fermentation broth retained 98% antifungal activity after thermal treatment at 100 °C. In addition, the antifungal activity of fermentation broth was not affected by trypsin and protease K. However, the fermentation broth could not endure both extreme acid and alkali treaments, and the suitable pH ranged from 4.0 to 10.0. The methanol extract from strain HN-Q-8 had significant inhibition effect on Rhizoctonia solani, with the two main active substances in it including fengycin and surfactin. [Conclusion] The fermentation broth of strain HN-Q-8 exhibited stability and strong antifungal activity, and has promising prospects for its further study, development and application.

Abstract:[Background] It was rare to compare the difference of endophytic bacterial communities in apple varieties thought many investigations had been reported on endophytic bacteria in apple trees recently. [Objective] By analyzing and comparing the diversity of endophytic bacterial communities in apple trees of 8 different varieties in Xinjiang and Kyrgyzstan, the abundant microbial resources would be explored. [Methods] Using MiSeq high-throughput sequencing method, sequences at V3?V4 regions of the 16S rRNA gene were amplified, then communities and functions of endophytic bacteria were analyzed in these apple varieties. [Results] valid sequences of 61 487?71 583 bp were obtained from varieties of apple trees, respectively, which clustered into 24?92 distinct operational taxonomic units (OTUs). Shannon index and Simpson index were 0.729?1.177 and 0.265?0.457, respectively. Species diversities of endophytic bacteria in Xinjiang apple varieties were richer than that in Kyrgyzstan varieties. The taxonomic analysis of endophytic bacteria showed that total OTUs of Proteobacteria and Actinobacteria covered 61.16%?97.08% of endophytic bacteria in different apple trees, respectively. They were the dominant bacteria phylum of apple trees. The number, composition and abundance of dominant genus were different in these varieties. OTUs in the genus Massilia and Arthrobacter were the richest, ranging from 6.06%?71.37% and 1.29%?17.86%, respectively. There were some microbial groups of beneficial functional characteristics related to promoting growth, stress resistance or degradation of toxic and harmful substances in the environment in these dominant bacteria. The function prediction showed that the functions of endophytic bacterial community in different apple varieties were different. The function informations of microbial community of Xinjiang varieties were more than that of Kyrgyzstan varieties, which included chemoheterotrophy, aerobic chemoheterotrophy, ureolysis, arsenate detoxification and dissimilatory arsenate reduction. In addition, a large number of unclassified genera were observed in these varieties, ranging from 13.74% to 69.60%. [Conclusion] The diversity of endophytic bacterial communities of these apple varieties were relatively rich, and there were significant differences in the composition and function. Meanwhile, it provided clues to mine new microbial resources and explore the function of endophytic bacterial communities in apple varieties.

Abstract:[Background] With the continuous development of bamboo rat breeding industry, the restriction of artificial breeding technology leads to the continuous occurrence of bacterial diseases, and Escherichia coli disease has become the focus of prevention and control. [Objective] The purpose of this study is to isolate the pathogens that cause the death of bamboo rats in a large-scale bamboo rat farm in Mianyang, Sichuan. Through the histopathological observation of pathological mice, genetic evolution analysis and drug resistance analysis of pathogenic bacteria provide case support for the prevention and treatment of bacterial diseases in bamboo rats. [Methods] Morphological observation and 16S rRNA gene sequencing analysis were used to identify pathogens, and histopathological observation, genetic evolution analysis, drug sensitivity test and drug resistance gene analysis were performed on the isolates. [Results] One pathogenic Escherichia coli isolated from the liver of bamboo rat pathological tissue sections showed severe lung, liver and kidney lesions, and the degree of spleen tissue lesions was not significant. Amikacin, gentamicin, spectinomycin, ampicillin, ceftazidime, cefepime, cefotaxime, polymyxin, tetracycline, doxycycline have good antibacterial effects on Escherichia coli, but the bacteria were not sensitive to conventional antibiotics such as levofloxacin, norfloxacin, enoxacin, neomycin, erythromycin, fluorfenicol, compound xinnomin. The strain carries aminoglycoside resistance genes (aac(3)-II, aph(3′)-II), chloramphenicol resistance genes (cmlA, floR). [Conclusion] The disease in the rodent farm was caused by pathogenic Escherichia coli. This pathogenic Escherichia coli strain carries resistant genes such as aac(3)-II, aph(3')-II, cmlA, floR.

Abstract:[Background] In June 2016, Takifugu bimaculatus, cultured in Fujian Seawater Fish Seedling Breeding Research Base, outbroke the skin ulceration disease. The main symptoms of diseased fishes were characterized by slow swimming, lack of appetite, ulceration of mouth, epidermis and fins, and severe hyperemia of kidneys and spleen. [Objective] Identify the pathogenic, and provide the technical support for effectively preventing and treating skin ulceration in T. bimaculatus farming. [Methods] The dominant strains were isolated from the kidney, spleen and muscle tissue of diseased fish. The strains were confirmed to be a pathogenic bacterium by artificial intramuscular injection. The strains were identified by morphological, physiological and biochemical characteristics analysis, 16S rRNA gene sequence analysis, phylogenetic tree construction, and drug sensitivity analysis. [Results] The dominant strain SBDFT-1# was isolated from spleen of diseased fish, and was confirmed to be pathogenic strain of the skin ulceration disease. Strain SBDFT-1# was identified as Vibrio harveyi, with the morphological indexes of Gram-negative, single-flagellate, short rod-shaped, and a cell size of 0.9×2.0 μm. The colony of strain SBDFT-1# was milky white and yellow on the 2216E and TCBS plate medium, respectively, which were both central bulge and neat-transparent edges. Strain SBDFT-1# was sensitive to 14 antibiotics including Oxacillin, Cefotaxime, Ofloxacin, Streptomycin, Co-trimoxazole and Polymyxin B; while strain SBDFT-1# was resistant to 9 antibiotics such as Ampicillin, Penicillin, Tetracycline and Medecamycin. [Conclusion] This is the first report of V. harveyi as a pathogenic strain, isolated from the diseased T. bimaculatus, while it is a common pathogen in the economical fish farming. The study has a positive significantly guiding for the prevention and treatment of skin ulceration of T. bimaculatus.

Abstract:[Background] Serine protease plays an important role in the biological control of Trichoderma spp. [Objective] This study provided theoretical support for the development of the protease biocontrol preparation and genetic modification by studying the serine protease S8/S53 superfamily gene information and biological functions of Trichoderma virens. [Methods] Twenty-three serine protease genes were identified from T. virens Gv29-8 genome by bioinformatics analysis. Four serine protease genes identified from Arthrobotrys oligospora ATCC 24927 genome were used as controls. The characteristics, protein structure, evolutionary status and function of 27 serine protease genes were predicted and analyzed. [Results] The 27 genes were significantly different in structure, and the encoded proteins had typical serine protease catalytic triad structure, belonging to the S8/S53 superfamily, which were divided into 6 subfamilies. The conserved domain of proteases in the same subfamily was similar in length, with high similarity and conservative sequences near the catalytic residues. Phylogenetic analysis showed that the serine proteases of the same subfamily were clustered into one group. [Conclusion] Some serine protease genes of T. virens and A. oligospora have strong similarities in structure and protein properties and close genetic relationship, both belonging to the S8_PCSK9_ProteinaseK_like subfamily. It is speculated that T. virens and A. oligospora have similar functions of serine protease, which can inhibit plant pathogenic fungi and degrade the body wall of nematodes.

Abstract:[Background] Due to excellent growth performances, clear genetic backgrounds and mature genetic manipulations, Escherichia coli typical strains are commonly selected for producing β-farnesene, but the yields of β-farnesene still cannot meet the needs of industrial production until now. [Objective] Combined mutagenesis breeding techniques were used to obtain β-farnesene producing strains with high yields. [Methods] Escherichia coli EC-16 was bred by combination mutation methods of atmospheric and room temperature plasma (ARTP) and ultraviolet (UV), through the first screening with isopentenyl pyrophosphate (IPP) screening plate as selection pressure and the following second screening step with flask fermentation culture, finally subjected to fermentation tank verification. The genetic stability of the highly productive mutant strains was carefully comparatively tested by continuous multi-generation cultured. [Results] A mutant strain named E. coli HVK-9 was obtained and the yield of β-farnesene reached up to 22.1 g/L, which was 168.74% higher than that of the original strain. [Conclusion] The combinational mutations by virtue of ARTP-UV with isopentenyl pyrophosphate resistance screening model in this study can significantly improve the positive mutation rates, which can finally confirm the effectively increase with the yield of β-farnesene. The mutant strain HVK-9 has good genetic stability as an industrial fermentation strain, which lays a good foundation for the industrial production and application of β-farnesene.

Abstract:[Background] Endophytic fungi is a reservoir of bioactive small-molecular natural compounds, but it is difficult to obtain structurally novel compounds and pronouncedly active analogues because of enormous species dispersed in nature, and repeated isolation became an bottleneck in the mining process. [Objective] The objective is to rapidly find new biologically active metabolites with combination of LC-MS, DNP and bioactivity evaluation. [Methods] Pure metabolite was isolated through silica gel and recrystallization. The structure was elucidated by spectroscopic and X-Ray techniques. The biological activity was evaluated through 96-well dilution method. [Results] Eleven endophytes were isolated and identified from B. balsamifera (L.) DC. A targeted endophyte identified as Diaporthe sp. was selected and an active compound Cytochalasin H was afforded from the rice extract of this fungus, its inhibitory effects against Bacillus subtilis was assayed with a MIC value of 32 μg/mL. [Conclusion] This study introduced a new extensive method in mining for bioactive secondary metabolites from endophytes of B. balsamifera (L.) DC., which provides references for rapidly exploration of effective lead agents.

Abstract:[Background] In recent years, thrombotic diseases have seriously affected people’s health even at younger age. The research of efficient, safe and specific thrombolytic drugs will be of great significance to human health. [Objective] To establish a method for separation and purification of plasmin produced by Ophiocordyceps sinensis from solid culture, and to analyze the enzymatic properties of purified plasmin. [Methods] Ophiocordyceps sinensis plasmin was separated by ammonium sulfate salting-out, HiTrap SP cation exchange chromatography and Superdex 75 gel filtration chromatography. The protein concentration was determined by Bradford method, the plasmin activity was determined by the fibrin plate method, the purity was determined by Native-PAGE, and the relative molecular weight was determined by SDS-PAGE. [Results] In solid culture, Ophiocordyceps sinensis mycelia could produce at least two plasmins, wherein the purified OSP-1 specific activity reached 4 186.25 U/mg, the purification factor was 41.69 times. OSP-1, a serine protease, is composed of two subunits with relative molecular weights of 27.60 kD and 23.83 kD respectively. The optimum temperature and pH of the enzyme were 40 °C and 4.0, respectively. Cu2+ promotes OSP-1 activity, while Zn2+ inhibits enzyme activity. It was also found that OSP-1 not only displayed the ability to degrade fibrin but also activated plasminogen. The enzyme sequentially degrades the γ chain, the Aα chain, and the Bβ chain in the process of hydrolyzing fibrin. [Conclusion] The above results provide a theoretical basis for the development of the enzyme into a new thrombolytic drug.

Abstract:[Background] HPS is the pathogen of Gl?sser’s disease. Due to the drug resistance caused by long-term use of antibiotics and the lack of interactive immune protection of inactivated vaccines, it’s impossible to effectively prevent and control Gl?sser’s disease at present. [Objective] The purpose of this study was to understand the serotypes, genotypes and drug resistance characteristics of HPS in Anhui province, and to prevent and control Gl?sser’s disease more effectively. [Methods] The serotypes of 44 strains of HPS, isolated in Anhui province from 2008 to 2017 were identified by polymerase chain reaction (PCR), the genotyping was carried out by MLST, and MIC was determined by microdilution method. [Results] A total of 6 serotypes (1, 4, 5, 7, 13 and 14) were detected in 44 strains of HPS, serotypes 4 and 13 were dominant serotypes, each accounting for 40.91%. A total of 9 ST types (ST241, ST267, ST268, ST269, ST270, ST271, ST272, ST273 and ST274) were detected in MLST classification, the dominant genotypes were ST267 and ST268, respectively accounting for 40.91%. Drug resistance analysis showed that the susceptibility rates of gentamycin (GEN) and penicillin (PEN) were 100% and 93.2%, respectively. There were 86.4% of the isolates showed multiple drug resistance, and there were 33 types of multi-drug resistance spectrum, among which the ratio of COT-TET was the largest (42.4%). [Conclusion] The epidemic serotypes and genotypes of HPS in Anhui province are diversified, with high genetic heterogeneity and severe multi-drug resistance. There was no significant correlation between serotype, ST type and drug resistance.

Abstract:As a bottom-up constructed microbial community, the synthetic microbial system has the characteristics of low complexity, controllability and operability compared with natural microbial communities. It is an emerging biotechnology and draws on synthetic biology, systems biology, biological evolution and other knowledge. Furthermore, by rational designing, planning and regulating, it becomes an experimental platform for studying microbial ecology theory, and a microbial system for validating known theories. In this paper, we first briefly introduce the concept of synthetic microbial system and its origin, expound its basic construction principles. We then introduce its ecological theoretical basis, and summarize its applications in recent years. Finally, we present our outlook on the development of synthetic microbial system, including the need to design and construct more complex synthetic microbial communities, and to optimize ecological models.

Abstract:Coronaviruses are the common pathogenic microorganisms that infect human and animals and cause health hazards. Cell immune responses are induced to fight against coronavirus infection in infected cells. In order to initiate transcription and translation and to assemble the next generation in infected cells, viruses respond to cellular immune response and participate in many cellular activities. When specific receptors such as death receptors are bound by viral proteins, cells initiate apoptotic processes. Some viral proteins play critical roles in promoting or inhibiting apoptosis in the apoptotic process. For example, S protein induces external apoptotic pathway by binding to death receptor in cell membrane, M and S proteins induce internal apoptotic pathway by causing endoplasmic reticulum stress and Ca2+ imbalance. On the other hand, E protein inhibits apoptosis in infected cells. This article reviews the mechanism of pro-apoptotic or anti-apoptotic effects of coronavirus on infected cells. By understanding the different roles of different viral proteins in extrinsic and intrinsic apoptotic pathways, it is expected to provide ideas for artificial intervention in cell regulation for prevention and control of coronavirus infection.

Abstract:Pichia pastoris is a widely used host for recombinant protein expression. As a basic tool for metabolic engineering, gene editing technology exhibits great importance for the metabolic modification of Pichia pastoris. In the past decade, gene editing technologies have developed rapidly. Many new techniques, such as ZFN, TALEN and CRISPR/Cas9, were developed besides homologous recombination and Cre/loxP recombination which makes gene editing in P. pastoris more convenient and efficient. This review summarized the principle, application and research progress of gene editing techniques in Pichia pastoris. This paper also prospected the future development of gene editing in P. pastoris based on the advances of related fields.

Abstract:The cyclodextrin glycosyltransferases (CGTases) are enzymes that used to synthesize cyclodextrin, and this method of production of cyclodextrin is mostly used at present. we described several engineering strains which were used to produce CGTase including Escherichia coli, Bacillus subtilis and Pichia pastoris, and the expression system of E. coli is one of the most widely used. In addition, we also summarized the effective strategies for high efficient expression of CGTase including appropriate expression vector, promoter and signal peptide, codon optimization, co-expression with molecular chaperone. The purpose of this review is to provide a reference for relevant research of CGTase.

Abstract:The coral reef ecosystem with extremely high value of ecology and economy are the most prominent and representative ecosystem in the tropical ocean. However, coral reef degradation caused by coral diseases has become one of the major threats of coral reef ecosystems. Many microbial pathogens (including bacteria, fungi, and virus) are thought to be closely related to coral disease. Identification of coral pathogens and establishing their rapid detection methods are crucial for epidemiological investigation and formulating of prevention and control measures of coral diseases. This paper mainly reviews the related microbial pathogens of coral diseases and research progress in molecular diagnosis techniques for coral pathogens.

Abstract:Since 2017, the Experimental Course of Microbiology, has been designed as one of the key courses for undergraduates in China Agricultural University, and was emphatically provided with financial and human support to promote its development and reformation and the improvement of quality of teaching/studying. This article introduced a series of reform measures we have taken in the teaching of Experimental Course of Microbiology, including application of staged teaching mode and “Fromm’s Expectation Theory”, instillation the concept of “aseptic operation” and enhancement its related technologies, refining the operation steps and implementation of “person-to-person” assessment, application of biostatistical mapping and information dissemination platforms, encouragement and tutoring undergraduates to participate in experimental competitions, etc. Our reform measures have achieved good results. In addition, we also put forward some specific suggestions for future class teaching of experimental teaching to promote its continuous development and improvement.

Abstract:Agricultural Microbiology is a specialized basic course in agricultural colleges and universities. With the rapid development of microbiology, the reform of traditional teaching concepts and methods is not only to meet the needs of the times, but also an inevitable trend of historical development. This paper explores the reform of the teaching process of agricultural microbiology from the following aspects: the reform of teaching materials, the expansion and extension of experimental teaching contents, and the change of teaching consciousness and ideas. The purpose of the reform is to better promote the cultivation of students’ innovative ability, so as to build a first-class socialist university with Chinese characteristics.

Abstract:[Background] The expression vector is an indispensable tool for genetic engineering. However, some of the fungus such as Trichoderma reesei lacks commercial expression vector and the traditional vector construction method is complicated and time-consuming, which makes protein expression difficult to carry out. [Objective] To generate a fast and novel method to construct the expression vectors based on the sequences of URA3 gene to generate a fungus’s universal expression system for research and development. [Methods] Based on the sequence of URA3 gene, we used the upstream gene fragment of URA3 promoter and downstream gene fragment of URA3 terminator to form homologous recombination arms. The promoter and terminator of URA3 was replaced with the promoter of CBH1 and the terminator of PDC during the construction process, respectively. The elements of the vector were linked seamlessly in one reaction using DNA topoisomerase I. Pcbh1-MCS-Tpcd, flanked by the homologous recombination arm of URA3, was assembled with other element fragments to form vector pTRUC. Red fluorescence protein, mCherry, was inserted into pTRUC which later was transformed into T. reesei to generate the final RUT-C30. Such expression system proves its feasibility via observation and determination of mCherry protein expression. [Results] After culturing the positive transformants on the PDA medium with 5-FOA, red fluorescence appeared on the hyphae tips and the septum. PCR result proved the expression of mCherry in the host. Western blotting analysis also showed the expression of mCherry. This method of vector construction is feasible as the constructed expression vector could express the recombinant protein. [Conclusion] The results demonstrate that the rapid construction of the expression system based on homologous recombination with the URA3 gene and the construction method is feasible. This will become a powerful tool to promote the expression of recombinant proteins in many eukaryotic expressing systems.

Abstract:[Background] West Nile fever is widely prevalent in the world, the risk of West Nile fever spreading to China has increased. The rapid detection method of West Nile virus (WNV) was studied to establish the method reserve for West Nile virus detection. [Objective] To establish a reverse transcription recombinase aided amplification (RT-RAA) assay for detection of WNV. [Methods] The primers and probes were designed based on the conserved genome sequence of WNV. An RT-RAA assay of WNV was constructed, and the reproducibility, specificity and sensitivity of the assay were evaluated. [Results] The RT-RAA assay had a constant temperature throughout the amplification reaction, the reaction temperature was 39 °C. The detection time was less than 20 minutes and the detection sensitivity could reach 10 copies. The method had excellent specificity as it didn’t have cross amplification with other arbovirus such as chikungunya virus, dengue virus, Japanese encephalitis virus and Yellow fever virus. The results of sample detection also meet the expectation. [Conclusion] The RT-RAA assay of WNV established in our study was rapid, specific and sensitive. It can be used for rapid detection and epidemiological surveillance of WNV at the port.