Abstract:[Background] Mangroves, trees and shrubs adaptive to salt, grow along the coast at tropical or subtropical latitudes. Mangrove endophytic fungi are the second largest community of marine fungi, which are rich in secondary metabolites with diverse chemical structures and excellent bioactivities. Therefore, mangrove fungi have proven to be an essential source of natural products and novel drug leads. [Objective] To study the diversity of culturable endophytic fungi from Zhanjiang mangroves and to identify bioactive secondary metabolites from the isolate Stemphyliums sp. SCSIO 40436. [Methods] Endophytic fungi isolated from Zhanjiang mangrove forests were identified based on internal transcribed spacer (ITS) sequence. The production of secondary metabolites on rice and oat solid media was evaluated by HPLC-DAD and filter paper diffusion method. The crude extract was subjected to column chromatography with silica gel and Sephadex LH-20, medium-pressure liquid chromatography, or semi-preparative RP-HPLC. The metabolite structures including absolute configuration were determined by spectroscopic methods (HRESIMS, NMR), X-ray crystallographic analysis, and comparison of electronic circular dichroism. Antimicrobial activities were measured against four indicate strains via the broth microdilution method. [Results] A total of 52 strains of culturable endophytic fungi were isolated from the Zhanjiang mangrove forests and categorized into 29 genera. The strain Stemphylium sp. SCSIO 40436 was found to be prolific in metabolites. Extensive chromatographic separation of the ethyl acetate extracts afforded five polyketides: stemphol (1), macrosporin (2), altersolanol A (3), alterporriol E (4), and alterporriol D (5). The structures of 1 and 3 were confirmed by X-ray crystallographic analysis. Stemphol (1) exhibited a good activity against the Gram-negative bacterium Acinetobacter baumannii ATCC 19606 with a minium inhibitory concentration (MIC) value of 8 μg/mL. [Conclusion] A total of 52 endophytic fungal strains are isolated from the Zhanjiang mangrove forests, among which several strains shared <95% similarity of ITS rDNA sequences, which enriches the diversity of mangrove endophytic fungi. Stemphol (1) exhibits a moderate activity against the Gram-negative bacterium A. baumannii.

Abstract:[Background] Quantitative microbial risk assessment (QMRA), a valuable tool for estimating the burden of disease due to the exposure to pathogenic microorganisms, has been widely used abroad. However, the application in China is in its infancy and there is a lack of data on human exposure in marine bathing beaches. [Objective] To collect exposure data of swimming populations and apply them in marine bathing beaches to assess the feasibility of fecal coliform as a risk assessment indicator. [Methods] The correlation of water quality and fecal coliform concentration in six marine bathing beaches with environmental factors was analyzed, and the exposure data of domestic swimming populations were collected based on questionnaire survey. Then QMRA was employed to evaluate the risk of gastrointestinal diseases from each marine bathing area. [Results] Fecal coliform concentration in the six bathing areas was significantly correlated (P<0.01) with water temperature, air temperature, and total cloud cover. The fecal contamination in the southern bathing areas was more serious than that in the northern bathing areas, and the 95th percentile of fecal coliform concentration was much higher than the threshold of the domestic "poor" water quality standard. The volume of seawater swallowed by children, male adults, and female adults in a single bathing event was 35.1 mL (95% confidence interval=32.4–37.8, α=0.578, β=0.016), 45 mL (95% confidence interval=31.1–59.3, α=0.532, β=0.012), and 35.7 mL (95% confidence interval=29.7–41.8, α=0.753, β=0.032), respectively. The risk of gastrointestinal diseases from all six marine bathing sites was well below the safety threshold set by the US Environmental Protection Agency. [Conclusion] It is recommended Enterococcus and human Bacteroides, rather than fecal coliform which can reflects fecal contamination, be used as indicators of human health risk from marine bathing sites.

Abstract:[Background] Triploid Populus tomentosa is suitable for the ecological and economic development of the Yellow River and serves as an important tree species for forestry extension projects in China. Endophytic bacteria of triploid P. tomentosa play a role in the disease prevention, growth promotion, nitrogen fixation, and biological repair. [Objective] The objective was to analyze the diversity of endophytic bacteria and fully explore the microbial resources in triploid P. tomentosa. [Methods] In this study, the diversity of endophytic bacterial community in the roots, stems and leaves of P. tomentosa from the research base of Beijing Forestry University in Guanxian county, Shandong province was analyzed via 16S rRNA gene high-throughput sequencing and plate streaking. The variation trends and rules of endophytic bacterial diversity in different tissues of triploid P. tomentosa were clarified to lay a theoretical foundation for the further application of endophytic bacteria. [Results] The endophytic bacteria of triploid P. tomentosa had the highest richness and diversity in the roots while the lowest in the leaves. Pseudomonas and Actinobacteria were the dominant phyla, and Burkholderia, Pseudonocardia, and Acidovorax were the dominant genera. The structure of endophytic bacterial community varied among different tissues. The functions of the endophytic bacteria mainly involved amino acid metabolism, vitamin metabolism, degradation of aromatic compounds, and glycolysis. A total of 217 endophytic bacterial strains were isolated, belonging to 44 species of 23 genera. Among them, four strains shared the 16S rRNA gene sequence similarity below 97.5%, which might be new taxa. [Conclusion] In conclusion, the diversity of endophytic bacteria in the roots, stems and leaves of triploid P. tomentosa was significant. The diversity and richness of endophytic bacteria were higher in the roots than in the stems than in the leaves. Some endophytic bacterial resources were obtained by traditional isolation and culture methods, including 4 strains with 16S rRNA gene sequence similarity less than 97.5%. However, the results of high-throughput sequencing showed that a large number of endophytic bacterial strains had not been cultured in this experiment. It was necessary to further develop new high-throughput isolation, culture and identification methods to fully explore more uncultured and difficult endophytic bacterial strains.

Abstract:[Background] With the development of pharmaceutical industry, an enormous amount of antibiotic fermentation residue is produced every year in China, which has caused environmental pollution and waste of resources. However, the residue is rich in proteins and other nutrients which can be used for secondary production, making it possible for effective utilization of the residue. [Objective] To screen yeast from oxytetracycline fermentation residue and optimize the culture conditions of the yeast in the medium with the residue as the main component for resource utilization of oxytetracycline fermentation residue. [Methods] Yeast strain was screened from oxytetracycline fermentation residue with the serial dilution-agar plating method. The strain was then identified through morphological observation and 18S rRNA sequence analysis. Single-factor experiment and Box-Behnken design were used to optimize the culture conditions and determine the optimal growth conditions of the strain in the medium with oxytetracycline fermentation residue as the main component. [Results] The Zygoascus hellenicus Y1 strain was screened out. The optimum culture conditions are as follows: oxytetracycline fermentation residue at 5%, glucose at 0.5%, inoculum at 2%, pH 5.0, 32 ℃, 160 r/min, 24 h. [Conclusion] Y1, which can make full use of the oxytetracycline fermentation residue for growth, was screened out, realizing the resource utilization of the residue and reducing the discharge of bacterial residue. The result lays a foundation for the preparation of yeast extract with oxytetracycline fermentation residue.

Abstract:[Background] Thermophilic group II introns are a class of retrotransposons that are composed of intron RNA and intron-encoded protein (IEP) and can move on chromosomes at high frequency under high temperature, and nowadays, they have been used to develop Themortargetron, an efficient gene-targeting tool. Therefore, it is highly important to elucidate their active catalytic sites for studying the "retrohoming" mechanism and developing new genetic tools. [Objective] To screen the key active sites from the domain of thermophilic group II intron Tel3c/4c-RT and obtain IEP mutants with inactivated reverse transcription function. [Methods] Firstly, bioinformatics method was used to analyze and screen the key amino acid sites that might affect the reverse transcription function of Tel3c/4c-RT. Then, site-directed mutations were performed on the selected key amino acid sites, and Thermotargetron plasmids were used to construct a targeting system of thermophilic group II intron with inactivated reverse transcription function. Finally, taking the lacZ gene of Escherichia coli as example, the targeting efficiency of the Thermotargetron mutant system was analyzed by blue-white screening, and the effect of the key active site of the Tel3c/4c-RT on the targeting efficiency of the thermophilic group II intron was verified in vivo. [Results] A total of 15 amino acid sites that may affect reverse transcriptional activity were screened out: D194, I195, S196, G197, C198, F199, Q241, G242, R274, Y275, A276, D277, D278, L324 and G325. The "retrohoming" of Tel3c/4c was almost completely lost when the G242 and R274 were mutated, suggesting that the G242 and R274 were the core catalytic sites affecting the reverse transcription function of Tel3c/4c-RT. [Conclusion] The core catalytic sites affecting the reverse transcription function of Tel3c/4c-RT were screened out, which laid a good foundation for in-depth study of the "retrohoming" mechanism of the thermophilic group II introns and the further development.

Abstract:[Background] Having special fragrance and various biological properties, linalool has become an important feedstock for food, pharmaceutical and cosmetics industries. With the development of synthetic biology, metabolic engineering of microorganisms has become an influential alternative for biosynthesis of linalool. However, the strong toxicity of linalool to host cells is a key bottleneck for microbial production of linalool and other monoterpenes. [Objective] This paper aimed to develop effective strategies for improving the tolerance of microbial host cells to linalool. [Methods] In this study, the ATP-binding cassette (ABC) transporters, reactive oxygen species (ROS)-related enzymes and transcription factors were overexpressed in Saccharomyces cerevisiae BY4741 to identify their roles in the tolerance of S. cerevisiae to linalool. In addition, adaptive laboratory evolution was adopted to obtain the S. cerevisiae strains with increased fitness towards linalool. [Results] Individual overexpression of ABC transporters (Yor1, Snq2, Pdr5, Pdr15 and Pdr18), ROS-related enzymes (Gre2, Ctt1, Yhb1, Gpx2, Trr1, Trx2 and Gsh2) and transcription factors (Ino2, Yap1, Yap5 and Stb5) in S. cerevisiae BY4741 failed to improve the tolerance of S. cerevisiae. Furthermore, S. cerevisiae with improved tolerance (lethal concentration of linalool was increased from 430 mg/L to 645 mg/L) were obtained via adaptive evolution and the SNV/InDel genes were analyzed by whole-genome resequencing. Mutations were found in YBR074W, YBR172C, YHR007C and YMR275C, which enhanced the tolerance to linalool. [Conclusion] The tolerance of S. cerevisiae to linalool was improved by evolutionary engineering, which laid a foundation for analyzing the mechanism of S. cerevisiae to tolerate monoterpenes and provided an excellent chassis cell for heterologous synthesis of monoterpenes.

Abstract:[Background] Umbelliferone, a compound of the coumarin family, demonstrates the medical value in a wide scope and is important raw material for industrial products. Due to the high cost of extraction from plants, more efficient methods for producing umbelliferone remain to be developed. [Objective] To heterogeneously produce umbelliferone by microorganisms. [Methods] In this study, the genes for the synthesis of coumarins were assembled from different plants. Saccharomyces cerevisiae was used as the expression host in which the metabolic pathway of tyrosine was modified. We then introduced the assembled genes to S. cerevisiae. [Results] The strain producing umbelliferone was successfully obtained, which showed the umbelliferone titer of (67.39±4.87) μg/mL. [Conclusion] The strains producing natural products of coumarins could be constructed via integration of the biosynthetic genes.

Abstract:[Background] Bioconversion via sucrose isomerase (PalI) is currently a preferred method to produce isomaltulose. However, this method still has some shortcomings, such as the formation of multiple products and the tedious purification process of PalI from cell extracts. [Objective] We sought to achieve efficient isomaltulose production via constructing a surface display strain for PalI on Y. lipolytica Po1g and reduce the proportion of by-products and purification cost. [Methods] The gene PalI encoding sucrose isomerase PalI from Klebsiella singaporensis LX3 was fused with the gene Pir1 encoding the anchoring protein Pir1 from the yeast cell wall by overlap extension PCR. The fused gene was then transferred into Y. lipolytica Po1g for the surface display of PalI. The enzymatic properties of the surface-displayed PalI were investigated via colorimetry with 3,5-dinitrosalicylic acid (DNS), and the products of sucrose conversion were detected by high performance liquid chromatography. [Results] The PalI surface display strain Pir1-PalI/Po1g was successfully constructed, and the highest activity of the displayed PalI reached (4 694.6±56.6) U/g of dry cell weight. The displayed PalI was stable at a broad range of 20-55 ℃ and pH 3.5-9.0, with the best performance at pH 6.0 and 45 ℃. Moreover, the displayed PalI led to significantly lower proportion of monosaccharide by-products during the bioconversion process than the free PalI. [Conclusion] Using Y. lipolytica Po1g as the host, we constructed a recombinant strain for the surface display of PalI, which provided a basis for the industrial production of isomaltulose.

Abstract:[Background] Staphylococcus aureus is a common zoonotic opportunistic pathogen. With the emergence of multidrug resistant strains, it is urgent to develop antimicrobial agents with different modes of action from antibiotics. [Objective] To isolate S. aureus phage and identify its functional lysin components as an effective specific antimicrobial agent. [Methods] The whole genome sequence of the phage was assembled and annotated for the mining of putative lysin encoding genes. Two putative lysin genes were respectively cloned into a prokaryotic expression vector. SDS-PAGE and Western blotting were employed to confirm the expression of the target proteins. We then verified the lytic activity by spotting the expression product on the host bacterial lawn. [Results] The isolated phage in this study could lyse its host bacterium and was named vB_Sau_P68. The phage genome was 139 409 bp with the GC content of 31.0% and encoded 220 open reading frames (ORFs). Under the transmission electron microscope, the phage appeared as an icosahedron with a contractile tail, which belonged to Myoviridae. Two putative lysin genes were annotated in the phage genome. ORF161 was predicted to encode lysin with a CHAP catalytic domain and ORF163 with a SH3_5 binding domain. The results of SDS-PAGE and Western blotting showed that Lys161 was expressed successfully and had lytic activity, while the expression of Lys163 was not be detected. The Lys161 sequence had no signal peptide or transmembrane region, and random coil was its major secondary structural element. [Conclusion] In this study, two lysin genes were cloned from a Staphylococcus aureus phage genome and expressed. The results suggested that the CHAP catalytic domain had lytic activity, while the SH3_5 binding domain was not expressed. The findings provide a theoretical basis for the exploration of the acting mechanism and application of lysin.

Abstract:[Background] Viruses in Lentinula edodes lie latent for long periods before the appearance of symptoms. However, once reactivated, they will cause great economic loss. [Objective] To analyze the mycovirus diversity in Chinese L. edodes core collections. [Methods] RT-PCR and RT-qPCR were used to detect 34 types of mycoviruses in 56 core collections of Chinese L. edodes. [Results] The target genes of 14 and 5 types of viruses in most of the positive strains showed bright and faint bands, respectively. The brightness of the bands was positively correlated with virus content. To be specific, the higher the virus content in the positive strain, the brighter the band. Among the 34 viruses, LeFV5 and LeMV1 were detected in all the 56 core collections. The detection rates of 8 viruses in 21 cultivated strains and 6 viruses in 35 wild strains were over 80% and 4 viruses (LeDFV2, LeFV2, LeSV, and LeHV2) were shared by the two. The cultivated strains and wild strains carried 8-21 and 9-23 viruses, respectively, with 16.4 and 16.7 on average. [Conclusion] L. edodes germplasms generally carry a variety of complex mycoviruses. The amplified bands have different brightness in positive strains. L. edodes germplasms with great genetic difference share some viruses with high detection rate, suggesting that these viruses arose early in the evolution of L. edodes.

Abstract:[Background] South China suffers from serious cadmium (Cd) pollution. Symbiosis with beneficial microorganisms can relieve Cd toxicity and improve crop resistance to Cd through direct or indirect mechanisms, and thereby promote crop growth. Cd-resistant growth-promoting bacterial inoculants have broad application prospects. [Objective] Growth-promoting bacteria that can resist Cd and promote soybean growth are screened from the roots or rhizosphere of Cd-contaminated plants in South China, which is expected to enrich beneficial microbial resources in soybean production. [Methods] Strains were isolated from the roots or rhizosphere of the plants with the streak plate method, and preliminarily identified through the analysis of physiological and biochemical characteristics and 16S rRNA gene sequence. Meanwhile, pot experiment was used to explore the effects of these strains on soybean growth under Cd stress. Malondialdehyde (MDA) content and total antioxidant capacity (T-AOC) were determined to explore Cd-resistant mechanism of these strains. [Results] Four strains D1, D2, D3, and D4 were isolated, which all can solubilize phosphate and produce indole-3-acetic acid (IAA) and siderophore. They belong to Acinetobacter, Microbacterium, Paenibacillus, and Providencia, respectively. The pot experiment on soybean Baxi 10 showed that these four strains had Cd-resistant capacity and could promote soybean growth. Shoot dry weight, root dry weight, and plant height of soybean inoculated with D4 were increased by 28%, 35% and 31%, respectively, in the absence of Cd. In the presence of 20 mg/kg-CdCl₂·5/2H₂O, shoot dry weight of soybean plants inoculated with D1, D2, D3, and D4 was raised by 35%, 55%, 53%, and 43%, respectively. The MDA content in the shoots of soybean plants inoculated with D2 and D4 was decreased by 23% and 29%, respectively. The T-AOC in the shoots of soybean plants inoculated with D1 and D4 was elevated by 11% and 13%, respectively. [Conclusion] The screened strains are expected to be developed into microbial fertilizers and applied into Cd-contaminated farmland for improvement of crop growth and yield. Meanwhile, it lays a theoretical basis for the study of Cd-resistant mechanisms of plant growth-promoting bacteria.

Abstract:[Background] The unique eco-environment in Qinghai province allows the growth of special microbial resources. [Objective] To explore Bacillus resources that can tolerate the plateau environment. [Methods] Antagonistic activity and indole-3-acetic acid (IAA) production of Bacillus atrophaeus CKL1 were determined by plate confrontation and chromogenic method, respectively. Moreover, the low-temperature tolerance and salt resistance of CKL1, as well as the influence of CKL1 on seed germination, seedling growth and the content of chlorophyll, proline, and malondialdehyde of Avena sativa 'Qingyan 1' under salt stress were detected. Then, the whole genome of CKL1 was analyzed by next-generation sequencing and the functional genes were dissected. [Results] CKL1 significantly antagonized Fusarium graminearum and F. acuminatum (inhibition zone diameter >15 mm). The reaction solution of CKL1 and Salkowski reagent turned red and the strain grew in LB medium with 13% NaCl and at 4 ℃, indicating that it could produce IAA and was tolerant to salt and low temperature. CKL1 significantly promoted seed germination and seedling growth of 'Qingyan 1' under salt stress, significantly increased the content of chlorophyll and proline, decreased the content of malondialdehyde, and enhanced the salt resistance of 'Qingyan 1'. The genome of CKL1 was 14 281 280 bp, and 3 303 functional genes were annotated against GO. The genome encodes genes related to the synthesis of lipopeptides iturin and surfactin, and the synthesis of IAA, gene clusters related to the synthesis of osmoregulation substances such as proline and betaine, and the Na⁺/H⁺ antiporter in stress response, as well as the key genes encoding transcriptional regulators involved in response to high salt and low temperature. [Conclusion] The study lays a theoretical basis for using Bacillus to promote the growth of A. sativa under salt stress.

Abstract:[Background] Lactic acid bacteria are widely distributed in human and animals. They are the main force to maintain the balance of gastrointestinal flora and improve immunity. In recent years, as antibiotics are banned to be used as feed supplements for animals, the incidence of animal diseases has been on the rise. An essential solution is to develop new feed supplements based on the characteristics of lactic acid bacteria and their active metabolites. [Objective] To isolate and screen lactic acid bacteria with excellent antibacterial properties from soil and to analyze and evaluate the characteristics of their active metabolites. [Methods] Two acid-producing strains with excellent antibacterial activity were screened with bromocresol purple plate method and Oxford cup method, which were named H-3 and H-4, respectively. After morphological identification and 16S rRNA gene sequencing, the growth curves and acid production of the two strains were detected, respectively. The effective components of antibacterial substances produced by the two strains were analyzed based on acid exclusion, protease treatment, and heat treatment. [Results] H-3 and H-4 were preliminarily identified as Pediococcus acidilactici, which demonstrated good growth performance and acid production capacity. The supernatant of the fermentation broth of the two strains showed obvious inhibitory effect on Escherichia coli, Staphylococcus aureus, Salmonella choleraesuis and Shigella flexneri. At pH 6.0, the supernatant maintained strong antibacterial activity, while at pH 7.4, the antibacterial activity of the supernatant of H-4 disappeared. The antibacterial activity of the supernatant treated with protease decreased or even disappeared, but the high-temperature treatment had little influence on the activity, suggesting the existence of a protein in the antibacterial substances with thermal stability. [Conclusion] H-3 and H-4 have broad-spectrum antibacterial activity, and in addition to organic acids, there is also a protein in the produced antibacterial substances. The result in this study lays a theoretical foundation for the research and development of new green feed supplements and antibiotic alternatives.

Abstract:[Background] With tenacious vitality, Stipagrostis pennata is a pioneer plant species in the desert, which features fast reproduction, high seed yield, and wide spread of seeds. [Objective] To test the growth-promoting endophytic bacteria of S. pennata. [Methods] The endophytic strain Z1 was isolated from the seeds of S. pennata and the biochemical characteristics, fermentation conditions, and growth-promoting performance were explored. [Results] The colonies of Z1 were spherical, yellow, and opaque, with small protrusions in the center and wrinkled and wet margins. Microscopy showed that it was straight rod-shaped with fine flagella on the surface and the size of (0.5-1.0) μm×(1.0-3.0) μm. The Gram staining test, indole test, oxidase test, starch hydrolysis test, V-P test, and gelatin liquefaction test all demonstrated positive results, while the methyl red test and urea hydrolysis test showed negative result. Moreover, it was identified as a Pantoea strain. Z1 produced Indole-3-acid, with the yield of 3.14 mg/L. Moreover, it can solubilize phosphorus and potassium and secrete iron carrier. The optimum fermentation conditions of Z1 are as follows: peptone as nitrogen source, soluble starch as carbon source, calcium carbonate as inorganic salt, and pH 9.0. The wheat growth-promoting effect of Z1 was obvious 10 days after the inoculation, as the leaf width, plant height, and root length were 60.0%, 13.5%, and 8.0% larger, respectively, and the leaf water content, total chlorophyll content, nitrogen content, and soluble protein of wheat seedlings were significantly increased (P<0.05). [Conclusion] Z1 is a functional bacterium with growth-promoting potential, which can be used for microbe-plant interaction and desertification control.

Abstract:[Background] Fusarium equiseti (Corda) Sacc. caused root rot is one of the principal soil-borne diseases that have resulted in a decline in the production and quality of Saposhnikovia divaricata in recent years. Because of environmental safety and no harm to humans and animals, biocontrol becomes an efficient approach for the prevention and control of plant diseases. [Objective] In order to mine the biocontrol strains with good antagonistic activity against F. equiseti in the rhizosphere soil of S. divaricata. [Methods] The dilution plate method was used to isolate bacteria from the rhizosphere soil. The antagonistic bacteria were identified via the filter paper method and the Oxford cup method. Antibiotic labeling was employed to mark antagonistic bacteria and assess their colonization capacity. Pot experiments were carried out to investigate their ability of mitigating root rot. The morphological, physiological and biochemical parameters, as well as the 16S rRNA gene sequence, were used to assess the taxonomic position. [Results] A total of 157 strains of bacteria were isolated from the healthy rhizosphere soil of S. divaricata, among which the antagonistic bacterial strain SC-119 with strong activity against F. equiseti was screened out. This strain showed an inhibition rate of 68.53% and exhibited strong colonization capacity and a broad antagonistic spectrum. In the pot experiments, SC-119 showed the control effect of 67.39%, which was 29.03%, 32.26%, and 16.13% higher than that of Trichoderma harzianum, Bacillus subtilis, and mancozeb, respectively. Finally, strain SC-119 was identified as Bacillus atrophaeus. [Conclusion] B. atrophaeus SC-119 has good antagonistic effect and biocontrol potential against F. equiseti, serving as an effective source for the biocontrol of root rot in S. divaricata. The findings of this study facilitate the development and utilization of strain SC-119.

Abstract:[Background] It remains a challenge to prevent and control Zanthoxylum bungeanum root rot in production, and the screening of biocontrol bacteria for the development of microbial agents seems to be a promising solution. [Objective] To analyze the genetic information of the antagonistic strain T-1, explore the root rot-antagonizing gene clusters, and reveal the antagonistic mechanism. [Methods] The methods of plate confrontation, morphological observation, physiological and biochemical index determination, and molecular biology were used to isolate and identify the antagonistic bacteria. The whole genome of the strain was sequenced, followed by sequence analysis and comparative genomics analysis. [Results] The strain was identified as Bacillus velezensis and numbered T-1. It inhibited 72% of the Fusarium solani, the pathogen of Z. bungeanum root rot, and hindered the growth of the front end of the mycelia. The results of in vitro antagonism experiments showed that T-1 had a wide range of antibacterial activities and had certain antagonistic effect on Z. bungeanum root pieces in vitro. Its whole-genome sequence data were submitted to SRA of NCBI to yield the accession number of SRX11086663. The genome was 3 886 726 bp, with GC content of 46.42% and 4 015 coding genes (89.74% of the genome). Comparative genomics analysis suggested that it had a high homology with the model strain B. velezensis FZB42, and the antagonistic gene cluster prediction indicated 12 gene clusters encoding the secondary metabolites in T-1 genome. Eight of them had functions known (butirosin A/butirosin B, macrolactin H, backland, fengycin, difficidin, bacillibactin, bacilysin, and surfactant), and the rest four had functions unknown. [Conclusion] This paper dissects the whole genome of B. velezensis T-1 and clarifies the gene clusters related to antagonism, which can serve as a reference for further research on the molecular antibacterial mechanism of this strain.

Abstract:[Background] The outbreak of corona virus disease 2019 (COVID-19) at the end of 2019 posed a major challenge to food safety. [Objective] To assess the contamination of Listeria monocytogenes in commercial fresh pork in Nanjing in the post-COVID-19 era. [Methods] We collected the fresh pork samples from different locations and in different packages and quarters during 2020-2021, and then analyzed the contamination rate and level of L. monocytogenes and the epidemiological characteristics of the isolates. [Results] The contamination rate of L. monocytogenes in fresh pork was 15.28% (77/504). The contamination rates of pork from open-air market and pork direct-sale stores were higher than that from supermarkets. Among different packaging methods, pre-package and simple package had higher contamination rate than bulk package. Further, the contamination rate varied significantly among different quarters, being the highest (27.78%) in the third quarter. Quantitative results demonstrated that 40.26% of the positive samples had the contamination level exceeding 10 MPN/g (MPN: most probable number). In particular, the contamination level of three samples exceeded 100 MPN/g. The results of serotyping showed that 1/2a-3a (48.05%) and 1/2c-3c (44.16%) were the main serotypes. Moreover, 19.50% of the isolates were multi-antibiotic resistant, and 2 isolates (2.60%) were sensitive to all the test antibiotics. Among the 77 isolates, 68 (88.30%), 46 (59.70%), and 45 (58.40%) were resistant to oxacillin, ampicillin, and cefotaxime, respectively. [Conclusion] In the post-COVID-19 era, the L. monocytogenes contamination of fresh pork varied among different locations, packaging methods, and quarters. A few products had high contamination levels, and the serotype distribution and resistance characteristics were diverse. Therefore, it is essential to strengthen food safety supervision to reduce the occurrence of foodborne diseases.

Abstract:[Background] Volvariella volvacea has high nutritional and medicinal value. Post-harvest quality deterioration of V. volvacea fruit bodies (VVFB) is mainly caused by the surface spoilage bacteria. There is no report on the surface bacteria of VVFB under fresh-keeping treatment. [Objective] To investigate the effect of ε-polylysine (ε-PL) combined with 1-methylcyclopropene (1-MCP) on surface bacteria of VVFB during storage. [Methods] VVFB were treated with ε-PL and 1-MCP and then stored. Through the plate culture method and 16S rRNA gene high-throughput sequencing, the surface bacteria of VVFB during storage were isolated and identified, and the dominant spoilage bacteria were determined. [Results] The plate culture method showed that the total number of colonies on the surface of VVFB increased during the storage. To be specific, the colony number of the treatment group (PC, ε-PL+1-MCP) was up to 7.16 lg (CFU/g) on the 6th day, smaller (P<0.01) than that 7.42 lg (CFU/g) in the control group (CK). A total of 16 and 19 strains were isolated from the PC group and CK group, respectively, which were dominated by Pseudomonas, Chryseobacterium, Bacillus, and Stenotrophomonas. The 16S rRNA gene high-throughput sequencing suggested 370 bacterial generas in 27 phylas were identified in the PC group, and 366 generas in 25 phylas were identified in the CK group. Among the strains, Stenotrophomonas had the highest relative abundance. [Conclusion] Stenotrophomonas is one of dominant spoilage bacterial genus of VVFB, and ε-PL combined with 1-MCP can effectively inhibit the growth of bacteria on the surface of VVFB.

Abstract:[background] At present, there are some difficulties in the diagnosis of Brucella canis. [Objective] To screen and analyze the specific epitopes of monoclonal antibody 4H3 against Brucella canis. [Methods] The phage display peptide library was used for screening. The 4H3 against B. canis was used as the target molecule, which was employed to coat the ELISA plate. Then, we applied 12-mer phage display peptide library for screening through 3 rounds of biopanning. In the biopanning, yield of phage increased from 5.00×10^-7 to 9.84×10^-6, and the false positive rate gradually decreased. We selected 14 of the screened positive clones for amplification, followed by extraction and sequencing of the genomic DNA. The affinity and specificity of the positive clones were detected by iELISA and cELISA. [Results] There were 3 different kinds of short peptide sequences in 14 monoclonal phages, which were KMSIRHPIRLPI, ILRRRRKRIIQI, and QRIHMRLTTQS, respectively. The affinity of the 3 short peptide sequences to monoclonal antibodies was in the order of KMSIRHPIRLPI>ILRRRRKRIIQI>QRIHMRLTTQS, and KMSIRHPIRLPI and ILRRRRKRIIQI had strong specificity. Thus, we further analyzed KMSIRHPIRLPI and ILRRRRKRIIQI and found that KMSIRHPIRLPI showed high similarity to peptidase C26 family protein (PuuD) of B. canis RM6/66 (75% similarity in amino acids) with 4 consecutive similar amino acids. ILRRRRKRIIQI had high similarity to 3-oxoyl-[acyl carrier protein] reductase (OAR) of B. canis RM6/66 (75% similarity in amino acids), with 2 consecutive similar amino acids. [Conclusion] Based on phage display peptide library, the short peptide sequences specifically binding to the monoclonal antibody against B. canis ZG were screened out.

Abstract:[Background] As an opportunistic pathogen, Gallibacterium anatis can cause poultry diseases such as ovaritis, salpingitis and peritonitis, which seriously threatens the development of poultry industry. [Objective] A shipment of chickens, died from a suspected G. anatis infection, was sent from a hennery in Sichuan. In order to explore the infection mechanism, prevention and treatment of G. anatis, we isolated and identified the bacterium and analyzed its whole genome sequence. [Methods] The bacterium was isolated and purified from the sample, followed by biochemical test, 16S rRNA gene sequence analysis, and drug sensitivity test. The whole-genome sequencing was carried out for species typing, annotation of virulence- and drug resistance-associated genes and phylogenetic analysis. [Results] The isolate was identified as G. anatis and named TS0001. Drug sensitivity test showed that the strain was sensitive to itrofurans and a few β-lactamines and resistant to some β-lactamines, chloramphenicol, some aminoglycosides, macrolides, tetracyclines and sulfonamides. The strain had a whole genome of 2 626 722 bp, and gene function annotation showed that it had strong self-modification capacity. A total of 83 genes related to virulence factors and drug resistance were annotated throughout the whole genome, and four prophage regions were predicted. The strain belonged to the sequence type ST69, and the phylogenetic tree constructed based on housekeeping genes demonstrated that it had the highest homology with the isolate 7990 from Mexican Gallus domesticus. [Conclusion] This study provides a reference for the research on the infection mechanism and prevention of G. anatis and enriches the molecular biological background for subsequent studies.

Abstract:[Background] Mycobacterium sp. LY-1 has become a dominant strain in industrial production because of its ability to metabolize natural phytosterols into important steroid drug intermediates. CRISPR/Cas9 as an efficient gene editing technology is the key to improving the yield and traits of industrial strains through metabolic engineering. However, due to the toxicity resulted from the high expression of Cas9 and the few available expression elements that have been reported in Mycobacterium, the moderate expression of Cas9 protein in Mycobacterium is greatly limited. [Objective] The endogenous expression elements were selected to activate the expression and reduce the toxicity of Cas9. [Methods] We used the online Berkeley Drosophila Genome Project (BDGP) to predict the endogenous expression elements from the transcriptome data of Mycobacterium genes in literature and available studies. The intensity of each expression element was assessed with enhanced green fluorescent protein as the reporter, and the expression of Cas9 protein was initiated with the expression elements of different intensities. [Results] Twenty-three expression elements with different expression intensities were obtained. The medium and weak expression elements reduced the toxicity of Cas9 to Mycobacterium sp. LY-1 and realized the moderate expression of Cas9 in the strain. [Conclusion] The endogenous expression element library of Mycobacterium sp. LY-1 was established, which laid a good foundation for the subsequent construction of CRISPR/Cas9 tools and the expression regulation of key enzymes in Mycobacterium.

Abstract:[Background] Lactobacillus acetotolerans is the dominant lactic acid bacteria species and plays an important role in Chinese liquor fermentation. L. acetotoleran G10, which was isolated from the fermented grains of sesame-flavor liquor, utilizes multiple carbon sources. [Objective] To analyze the mechanism for the multiple carbon source utilization of G10 based on whole genome sequencing. [Methods] The whole genome of G10 was sequenced by Oxford Nanopore Technologies, a third-generation platform for the sequencing of native DNA strands. Circlator was employed to circularize genome assemblies and Prodigal to predict genes and annotate protein-coding genes. bacterial pan genome analysis tool (BPGA) was used for pan-genome analysis. [Results] G10 was able to utilize 22 sugars and their derivatives. The genome size of G10 was 1 627 828 bp with 1 878 coding genes. G10 contained 292 genes related to carbohydrate metabolism, as annotated by Koyto Encyclopedia of Genes and Genomes (KEGG) annotation, and 44 genes related to Carbohydrate-Active EnZymes (CAZy) according to the CAZy annotation. Compared with other L. acetotolerans from food fermentations, G10 had the smallest genome with the highest numbers of total genes and genes related to starch and sucrose metabolism, and contained 426 unique genes. Compared with Lactiplantibacillus plantarum subsp. plantarum ATCC 14917^T, Limosilactobacillus fermentum ATCC 14931^T, Lacticaseibacillus casei ATCC 393^T, Levilactobacillus brevis ATCC 14869^T and Lentilactobacillus buchneri ATCC 4005^T from food fermentations. The genome of G10 was the smallest. The number of genes related to carbohydrate metabolism accounted for the highest proportion of total genes in G10. It had unique genes such as glvA, malP and glvC. [Conclusion] G10 can use a variety of carbon sources and adapt to various fermentation environments. The analysis of genomic information lays a genetic basis for further illustration of the fermentation performance of L. acetotolerans.

Abstract:[Background] Endophytic Streptomyces sp. SAT1, isolated from the roots of the medicinal plant Adenophora trachelioides, exhibits strong inhibitory activity against plant pathogenic fungi and bacteria, which has great biocontrol potential in agriculture and forestry. [Objective] The inhibitory effect of SAT1 and the types of active substances against bacteria in different media were revealed to provide theoretical basis and technical support for its biocontrol application. [Methods] The effects of medium components on the biosynthesis of antibacterial metabolites were investigated by measuring the antibacterial activities of fermentation broth and the mycelium extracts. Media with high or no inhibitory activities were selected for fermentation, and transcriptome sequencing of the mycelium was performed to analyze the function of differentially expressed genes. Moreover, ultraviolet (UV) spectrum and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) were used to identify the components of active substances. [Results] After fermentation with 7 commonly used media for Streptomyces, the fermentation broth and the mycelium extract from TSB, GS and R5 media did not inhibit the bacteria, while those from PDB, ISP2, H and MS media had strong antibacterial activity. Transcriptome sequencing analysis of PDB, ISP2 and TSB fermentation bacteria revealed a total of 3 567 differentially expressed genes. Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis demonstrated that most differentially expressed genes were enriched in metabolic pathways such as global and overview maps, amino acid metabolism and carbohydrate metabolism. As compared with TSB, 18 and 5 up-regulated genes were located in the biosynthetic gene cluster of moenomycin in PDB and ISP2, respectively. With the standard as the control, the main antibacterial substance was identified as moenomycin by UV spectrum and UPLC-MS/MS. [Conclusion] The biosynthesis of active secondary metabolites in SAT1 against bacteria could be increased by optimizing fermentation medium, with moenomycin analogues as the main active substance.

Abstract:[Background] Cladosporium sp. SYC63 is a potential biocontrol strain with mycoparasitism and antimicrobial activity. Little is known about the whole genome sequence of this strain, which limits its development and utilization. Genome sequencing and analysis will help us understand the mycoparasitism mechanism of this strain. [Objective] The aim is to analyze the genome sequence of SYC63 and preliminarily explore the mechanism of its mycoparasitism. [Methods] The whole genome of SYC63 was sequenced on the high-throughput sequencing platform, and the genomics tools were employed for sequence assembly, gene prediction and functional annotation, prediction of secondary metabolite synthesis gene clusters, and statistical analysis of carbohydrate-active enzyme genes related to mycoparasitism. [Results] A total of 17 contigs were obtained after genome assembly, with the total length of 31 912 211 bp, the GC content of 52.80%, and 12 327 coding genes. Among them, 4 029, 949 and 6 595 genes were annotated in KEGG, COG and GO databases, respectively. At the same time, 25 gene clusters for secondary metabolite synthesis were predicted. The strain SYC63 had more glycoside hydrolase and glycolipase genes than other mycoparasitic strains (Pestalotiopsis sp., Trichoderma sp. and Coniothyrium minitans). After treatment of the spore wall with rust fungus, the expression of cell wall-degrading enzyme genes was significantly up-regulated, which revealed that the mycoparasitism mechanism of SYC63 was different from that of Trichoderma. [Conclusion] We explored the mycoparasitism mechanism of Cladosporium at the genome level, providing reference information for further studying the mycoparasitism mechanism and mining the secondary metabolites, which is of great significance for the subsequent development and utilization of Cladosporium.

Abstract:[Background] Characterizing the protease activity is essential for revealing the potential ecological roles, industrial values, and pathogenic mechanism of Chryseobacterium. [Objective] This paper aims to analyze the genome sequence, characterize the protease, and optimize the enzyme production conditions of a novel Chryseobacterium strain, thereby providing the data support for subsequent research. [Methods] The genome sequence of the strain was obtained by high-throughput sequencing technology, and the conditions of protease activity and enzyme production were optimized by single factor tests. [Results] A strain, Chryseobacterium sp. ZHDP1, was isolated in this work. The genome of this strain had a length of 4 917 748 bp and the GC content of 35.95%. The average nucleotide identity (ANI) and DNA-DNA hybridization (DDH) indexes of the genome were 91.39 and 47.8, respectively. More than 20 protease genes were identified in the genome, and protease activity was detected in the supernatant of the fermentation broth. The protease had the optimum performance at 50 ℃ and pH 7.0 and was strongly inhibited by Zn²⁺, Mn²⁺, and Cr₂O₇^2-. The optimal conditions for the protease production of Chryseobacterium sp. ZHDP1 were as follows: culture temperature of 35 ℃, culture time of 35 h, inoculum amount of 4%, carbon source and nitrogen source of corn flour, and shaking speed of 100 r/min. [Conclusion] This study reveals the full-length genome sequence and the protease characteristics of Chryseobacterium sp. ZHDP1, which lays a foundation for the in-depth study of this strain.

Abstract:[Background] The commonly known poly-γ-glutamic acid (γ-PGA) producers are Bacillus subtilis, B. amyloliquefaciens, and B. licheniformis, while little is known about B. siamensis. [Objective] To study liquid fermentation conditions for γ-PGA production by B. siamensis. [Methods] B. siamensis CAU83, isolated by our laboratory, was used to produce γ-PGA by liquid fermentation. The effects of carbon sources, nitrogen sources, precursors, temperature, and pH on the synthesis of γ-PGA in shake flasks were investigated by single factor test and orthogonal design. [Results] The optimal carbon source, nitrogen source, and precursor for the synthesis of γ-PGA were 30 g/L lactose, 5 g/L yeast extract, and 60 g/L L-sodium glutamate, respectively. The optimal fermentation conditions were 37 ℃ and pH 7.0. The yield of γ-PGA increased by 260% from 8.4 g/L before optimization to 30.1 g/L after optimization. The fed-batch fermentation showed the peak yield (59.5 g/L) of γ-PGA at the time point of 60 h, with a productivity of 0.99 g/(L·h), which increased by 98% compared with the yield in shake flasks. The produced γ-PGA had a molecular weight of 3.8×10⁶ Da and a high polymerization degree. [Conclusion] The optimal fermentation conditions for B. siamensis CAU83 producing γ-PGA determined in this study provide a basis for the industrial production and application of this strain.

Abstract:[Background] Fusobacterium nucleatum, an opportunistic pathogen causing infectious diseases, is a risk factor for the occurrence of colorectal cancer. Simple and rapid techniques are urgently needed for the detection of F. nucleatum in clinical practice. [Objective] In this study, we established a direct observation and counting method to count F. nucleatum cells in samples with magnetic nanoparticle (MNP) probe under dark-field microscopy. [Methods] We prepared the MNP probe by modifying MNPs with the homemade polyclonal antibodies against F. nucleatum, which can bind to F. nucleatum specifically. Furthermore, we compared the sensitivity of our method with that of real-time fluorescence quantitative PCR (qPCR) for detection of F. nucleatum. [Results] The method established in this study showed the limit of detection as low as 3.42×10¹ copies/μL and the sensitivity 5 times higher than that of qPCR. For detection of the real samples, the results of ounting F. nucleatum by our method are consistent with that by qPCR. [Conclusion] The established method is simple, rapid (within about 30 min), sensitive, and economical for detecting F. nucleatum, which has the potential to serve the detection of clinical samples.

Abstract:[Background] Enterohemorrhagic Escherichia coli (EHEC) O157:H7 and enteropathogenic E. coli (EPEC) O55:H7 are common food-borne pathogens, causing intestinal infection and other diseases. The specific bacteriophages are of great potential for the development of new antibacterial agents. [Objective] To isolate the phages of O157:H7 and O55:H7 and to characterize their biological and genomic characteristics for future development of phage therapy. [Methods] Phages were separated from environmental water samples by the double agar overlay plaque assay. Their biological characteristics, including morphology, multiplicity of infection (MOI), host range, as well as one-step growth curve, were studied. Their genomes were sequenced by the Illumina MiSeq and then analyzed with RAST, Prokka, BLASTp. [Results] We isolated two potent phages of Myoviridae family, vB_EcoM_P251 and vB_EcoM_P255, using E. coli O157:H7 and O55:H7 as host, respectively. Their optimal MOI were both 1, and 91.9% and 90.8% of them adsorbed to the respective host within 15 min. Their lysis activity was high and stable at 37–60 ℃ and pH 4.0–11.0. P251 was only infectious to E. coli O157:H7 and O78:H11, while P255 was infectious to 11 EHEC and EPEC strains, including O55:H7 and O157:H7. The P251 genome and P255 genome are 136 254 bp and 111 068 bp in length separately, with GC content of 37% and 35%, respectively. Their genomes contain 227 and 173 open reading frames (ORFs), separately, 80 and 73 of which share significant similarities to functional genes. Besides, P251 contains 2 tRNAs. In addition, genomes of P251 and P255 share 72.24% nucleotide identity over 48% of their length. [Conclusion] Two new O157:H7 and O55:H7 phages, P251 and P255, with strong lysis activity, wide host range, as well as a great application potential in food-borne pathogenic E. coli control, were isolated and identified.

Abstract:[Background] Natural Cordyceps militaris is the fruiting body formed by Cordyceps militaris infects insect pupae or insects and has crucial biological and pharmacological activities. At the moment, the genome of C. militaris has been sequenced, but the research in molecular biology is limited. [Objective] To construct the Agrobacterium tumefaciens-mediated transformation (ATMT) system in C. militaris with uridine/uracil auxotrophic gene as the selection marker. [Methods] Orotidine-5'-monophosphate (OMP) decarboxylase is an essential enzyme for uracil synthesis. By using ATMT, We first knocked out the gene encoding pyrG of OMP decarboxylase in wild C. militaris by homologous recombination, and then constructed the uridine/uracil auxotrophic mutant. Afterward, the pyrG-carrying binary vector of Aspergillus oryzae was used to construct the complementary strain by ATMT. [Results] The pyrG was successfully knocked by homologous recombination and the uridine/uracil auxotrophic mutant was constructed. Under this background, the transformation efficiency reached (75±35)/10⁶ conidia after co-culture for 66 h at 22 ℃. In addition, the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase promoter PgpdA and α-amylase promoter PamyB, which are commonly used by filamentous fungi, failed to drive the expression of GFP or DsRed reporter gene in C. militaris, but the promoter IF of C. militaris extension factor could well express GFP. [Conclusion] We constructed an ATMT system with uracil auxotrophic as a selection marker, providing a promising genetic tool for research on recombinant expression and gene function of C. militaris.

Abstract:The recovery of phosphorus (Pi) from sewage has become a main direction of sewage reclamation. The polyphosphate-accumulating organisms (PAOs)-biofilm system, which features low carbon source consumption, simple procedure, high efficiency, and sludge reduction, has become a research hotspot in Pi recovery. In this paper, we summarized the basic principles of the system, the functional microorganisms and their metabolisms, and compared the different recovery modes and the performances. In addition, we summed up the research methods and technologies for microorganisms related to the system and their metabolic mechanisms. After describing the status quo of the system in Pi recovery, we proposed the application prospects of this technology, hoping to provide support for the basic research and technological development of PAOs-biofilm system.

Abstract:Grifola frondosa is a precious edible and medicinal fungus with anti-tumor, immunomodulatory, hypoglycemic, and antiviral activities. Polysaccharides are the main active component of G. frondosa, with the biological activity associated with the structure. We reviewed the structural characterization of active polysaccharides from G. frondosa. Some studies suggest that the blood sugar-lowering activity might be related to the chemical structure with β-1,6-glucan as the main chain, and the polysaccharides have better antitumor activity when the main chain is β-1,6-glucan or β-1,3-glucan. However, the complex structure makes it difficult to dissect the fine structure of G. frondosa polysaccharides. As a result, the structural characterization of G. frondosa polysaccharides is generally limited to monosaccharide composition, molecular weight, glycosidic bond type, branching structure, and rough molecular chain conformation. The advancing 2D-NMR and high-resolution mass spectrometry will help to disclose the structure-activity relationship and provide a theoretical basis for the development and utilization of G. frondosa polysaccharides.

Abstract:Increasing fungi have been developed as hosts of heterologous expressionfor the production of pharmaceutical proteins and enzymes in the last two decades. With the deepening research on the heterologous expression systems of fungi, people have gradually understood that the fungal N-glycosylation system is visibly different from that of higher animals, which has become a technological obstacle for fungi to produce higher animal-derived glycoproteins. This paper reviews the research progress of fungal N-glycosylation system in heterologous expression. Specifically, we introduced the detection techniques and transformation strategies of N-glycosylation in fungi, and compared the N-glycosylation system between fungi and higher animals, aiming to provide reference for the animalization and even humanization of fungal N-glycosylation system in the future.

Abstract:Sulfate-reducing bacteria (SRB), the anaerobic bacteria omnipresent in petroleum reservoirs, play an essential part in the cycling of sulfur in oil reservoirs. Many SRB can reduce sulfate to hydrogen sulfide which corrodes metal pipes, thus leading to lots of safety problems such as oil spill and causing economic loss of over 700 billion CNY each year. In this paper, the diversity of microbial communities living within biofilms of reservoirs and the synergistic corrosion mechanism of SRB with other related groups in biofilms were first summarized. Then, the sulfur-nitrogen-hydrogen biogeochemical cycle mediated by SRB in high-temperature reservoirs, the extracellular electron transfer mechanism, and the corrosion were discussed. Moreover, we introduced the field cases of SRB corrosion in high-temperature oil reservoirs to further elucidate the mechanism of SRB corrosion. Finally, we proposed to control the corrosion of SRB in biofilms in high-temperature reservoirs with nanomaterials.

Abstract:The rapid detection of microbial count has always been an issue to be solved in industrial production and food industry. Adenosine triphosphate (ATP) bioluminescence method with simple operation and short detection period can meet the needs of general microbial detection. However, this method still has some limitations, such as the high limit of detection, the influence of microorganisms and other factors (non-microbial ATP, extractants, luciferase activity, etc.), all of which impact the detection results. This paper first introduced the common methods for detecting the microbial count and reviewed the development history and principle of ATP bioluminescence; expounded the effects of non-microbial ATP, free ATP, microbial biomass, ATP extractants, and luciferase on the sensitivity and stability of ATP bioluminescence method. Furthermore, summarized the application status of this method in food industry, medical services, and sewage treatment. Finally, we discussed the optimization of the detection system and the application of ATP online detection. Through this review, we aim to provide a new idea for the efficient application of ATP bioluminescence method.

Abstract:Plant pathogens Rhizoctonia spp., which include diverse organisms, are soil inhabitants. They produce no spores and survive as hyphae and sclerotia. This paper reviewed classifications (according to number of nuclei, anastomosis, sexual reproduction, phylogeny, ect.) of Rhizoctonia fungi and analyzed the status quo of the taxonomy. Based on the number of hyphal nuclei, Rhizoctonia spp. are classified into uninucleate (UNR), binucleate (BNR), and multinucleate fungi (MNR). BNR and MNR are omnipresent, while UNR are rarely found in the nature. According to the result of anastomosis reactions, MNR and BNR are respectively categorized into 13 and 18 anastomosis groups (AGs). Some AGs are further divided into subgroups based on some stable characteristics, but the standards for the subclassification are not uniform. The results of molecular phylogenetic research also support the classification of AGs and subgroups. Based on the morphological characteristics of some Rhizoctonia isolates with sexual reproduction, MNR and BNR are identified as Thanatephorus and Ceratobasidium, respectively. At present, genome sequencing has been completed for at least 17 isolates of 9 AGs or subgroups which are important plant pathogens and orchid mycorrhizal fungi. Comparative genomics and mitogenomics have played an important role in the classification and phylogenetic research of Rhizoctonia. In summary, the classification systems of Rhizoctonia are special and complex. The authors also analyzed the problems in the taxonomy of Rhizoctonia and the future research trend, hoping to provide a reference for the future research on Rhizoctonia.

Abstract:Toxin-antitoxin (TA) system is prevalent in chromosomes and mobile genetic elements of bacteria and archaea. TA systems are diverse in structure and function, which are currently classified into eight types (type I-VIII). They are involved in biofilm formation, virulence, drug-resistant infection of host bacteria, regulation of plasmid copy number, and maintenance of prophage after its excision. In this paper, we reviewed the latest classification and functions of TA systems and the regulatory functions of antitoxin, and then briefly described the application of TA systems.

Abstract:Enterovirus D68 (EV-D68), a member of Picornaviridae, has emerged over the recent years, with large outbreaks worldwide. However, no specific vaccines and drugs against EV-D68 are available. Accumulating studies are extending our understanding on the pathogenesis of EV-D68. In this review, we summarized the research on EV-D68 receptors, hoping to provide a reference for the development of targeted antiviral drugs.

Abstract:The intestinal epithelium is the mucosal interface consisting of intestinal epithelial cells and their secretions. Owing to the progress of technology and the increasing attention to the role of intestinal microbiome, researchers have deepened the understanding of interaction between intestinal epithelium and microbiome. The available studies have demonstrated that intestinal epithelium regulates and maintains the colonization and distribution of microbiome, while the microbiome affects multiple barrier functions of the epithelium. They interact through a series of cellular and molecular mechanisms, maintaining intestinal homeostasis together. Moreover, their co-metabolites produced in the process can reflect the physiological or pathological state of the host and be used as biomarkers for diagnosis of diseases, evaluation of therapeutic effect, and prediction of prognosis. This paper reviews the research progress in the interaction between intestinal epithelial and microbiome and the underlying cellular and molecular mechanisms, which provides a theoretical basis for further research and clinical application. Finally, we predict the possible directions of the future research.

Abstract:Curriculum ideological and political education (CIPE) joins ideology and politics in curriculum to explore the effective ways of integrating knowledge transfer, ability training, and value shaping. The higher education has entered a new era of comprehensive CIPE. Microbiology has a wide professional and application scope, and it is closely related to the development of human society, production practice, and daily life. Owing to the rich ideological and political elements, Microbiology is an excellent carrier for conducting CIPE. Our microbiology teaching team has always been adhering to the educational philosophy of imparting knowledge and educating people, and is committed to the innovative reform of microbiology teaching. On this basis, the thinking and practice of CIPE have been carried out in recent years. This article summarizes the necessity, integration path, teaching evaluation, reflection and continuous improvement of developing CIPE in Microbiology, aiming to provide a reference for the reform of CIPE in colleges and universities.

Abstract:The application of the "five-stage" flipped classroom teaching model assisted by Blackboard platform in experimental teaching was explored based on the characteristics of research experimental teaching and related theories of blended teaching. The Microbiology Experimental Technology course was used as an example to expound the design thought of the teaching model, i.e., independent learning and experimental design before class, student discussion in class, implementation of open experiment, class presentation, and summary after class. The teaching effects of this model and existing problems were also discussed. The findings may provide reference for the application of flipped classroom teaching model in practical courses, in hope of cultivating students' scientific thinking and innovation ability.

Abstract:[Background] Microbiology is embracing a crucial period for development. Master's thesis, as the main output of graduate students, reflects the research trend in this discipline. However, previous bibliometric analysis mainly focused on papers from Web of Science and China National Knowledge Infrastructure (CNKI) rather than the theses from master's thesis databases. [Objective] Our aim in this paper is to explore the research approach and hotspots of postgraduate dissertations in the field of microbiology in China. [Methods] We analyzed 237 562 master's theses on microbiology in China Doctoral Dissertations/Master's Theses Full-text Database (CDMD) from 1999 to 2018 with the bibliometrics method. [Results] We found that the total number of microbiology-related theses in China showed an increasing trend from 1999 to 2018, and Zhejiang University, University of Chinese Academy of Sciences, and Nanjing Agricultural University ranked the top three in the number of published theses. According to the network analysis of keywords, research fields of postgraduates in China are diversified in the 10 years. The research hotspots in microbiology, such as environment, animal medicine, and human medicine, are also international research focuses, but basic microbiology is yet to be further emphasized. Some emerging topics such as metagenomics, proteomics and engineering, and medicine should be highlighted among postgraduates. [Conclusion] The result is expected to serve as a reference for topic selection by graduate students and their supervisors, and education and practice of postgraduates on microbiology in China.