Abstract:[Background] In recent years, the expansion of mariculture has caused the massive accumulation of excrement and residual feed, which has increased nitrogen and phosphorus content in aquaculture waters, aggravating eutrophication and causing harm to the environment. [Objective] To screen out the aerobic denitrifying phosphorus-accumulating strains from mangrove constructed wetlands, determine the optimal nitrogen and phosphorus removal efficiency of each strain, and then optimize the strain combination by response surface methodology to further improve pollutant removal. [Methods] From the 5 strains of salt-tolerant heterotrophic nitrifying-aerobic denitrifying bacteria isolated in our previous study, the aerobic denitrifying phosphorus-accumulating bacteria were screened by metachromatic granules staining and poly-β-hydroxybutyric acid (PHB) staining. The optimal nitrogen and phosphorus removal conditions of each strain were determined by single factor test. Box-Benhnken design was performed in Design-Expert to optimize the strain combination. [Results] Three strains of salt-tolerant aerobic denitrifying phosphorus-accumulating bacteria were screened out, which were Achromobacter pulmonis strain E43, Achromobacter xylosoxidans strain J1, and Pseudomonas oleovorans strain F2, respectively. E43 was found for the first time to have the function of phosphorus accumulation. The optimal strain combination was determined as E43:J1:F2=1:1:4. The average removal rates of ammonia nitrogen (NH₄⁺-N) and phosphate (PO₄^3--P) by this combination were 91.28% and 93.03%, respectively. [Conclusion] The bacterial strain combination improved the average removal rate of PO₄^3--P, which provided a preliminary basis for the subsequent application in the actual treatment of saline wastewater. This study is an effective attempt for the assembly of aerobic denitrifying phosphorus-accumulating bacteria and provides a feasible method for the assembly of efficient functional bacteria.

Abstract:[Background] Quorum sensing inhibitor (QSI), promising alternatives to antibiotics, can reduce the infectivity and virulence of pathogenic bacteria by interrupting quorum sensing pathways. Desert soils contain rich actinomycetes, which are an important source for the mining of QSI. [Objective] To investigate the bacterial diversity in the soil from Kumutag Desert and screen out the actinomycetes with the quorum sensing inhibitory activity. [Methods] Illumina NovaSeq high-throughput sequencing was employed to reveal the composition of bacterial community in the soil of Kumtag Desert, and the culture method to isolate the actinomycetes. The Chromobacterium violaceum 026 model was used for the screening of the actinomycetes with quorum sensing inhibitory activity. The functions of the strains screened out were initially evaluated. [Results] The soil samples harbored 150 genera of bacteria belonging to 96 orders of 23 phyla. The dominant phyla were Proteobacteria (61%) and Actinobacteria (28%). As for Actinobacteria, the genus with the highest relative abundance was Mycobacterium (87.3%), followed by Rhodococcus (6.8%) and Cutibacterium (0.9%). A total of 108 isolates were obtained and identified as actinomycetes belonging to 10 genera of nine families, in which the dominant genus was Streptomyces (65.76%). Thirteen strains with quorum sensing inhibitory activity were screened out, including 10 strains of Streptomyces and each one strain of Nocardiopsis, Bailinhaonella, and Kibdelosporangium. Strain Streptomyces sp. D67 exhibited broad-spectrum antimicrobial activities. [Conclusion] The bacteria in the soil of Kumutag Desert have high diversity and contain multiple strains of actinomycetes with quorum sensing inhibitory activity. Several strains with quorum sensing inhibitory activity demonstrate strong antimicrobial activity, which provide strain resources for the subsequent development of natural QSI and antimicrobial agents and lay a theoretical foundation for the development of biopharmaceuticals and biocontrol.

Abstract:[Background] The fungi in marine sediments are rich in bioactive natural products, while little is known about the fungi in coral reef sand and their natural products. [Objective] To isolate fungi from coral reef sand and separate their natural products, explore the fungal diversity, and lay a foundation for the development of natural products from marine fungi. [Methods] The fungi from coral reef sand were isolated with the dilution-plate method and identified based on the ITS rDNA sequences. The natural products of Cladosporium sp. GXIMD02067 were separated by silica gel column, reversed phase column, and preparative HPLC. The chemical structures of natural products were identified by nuclear magnetic resonance spectroscopy and comparison with literature data. [Results] Nineteen fungal strains belonging to 6 genera, 4 families, 4 orders, and 1 class were isolated, including 7 strains of Aspergillus, 6 strains of Penicillium, 2 strains of Cladosporium, 1 strain of Lecanicillium, 2 strains of Lulworthia, and 1 strain of Parengyodontium. GXIMD02065 and GXIMD02066 shared the ITS rDNA sequence similarity less than 87%, which indicated that they may be new strains. Seven compounds were separated from Cladosporium sp. GXIMD02067 and identified as pyrenocine A (1), pyrenocine B (2), thymidine (3), 1H-indole-3-carbaldehyde (4), p-hydroxybenzoic acid (5), methyl 2-(4-hydroxyphenyl)acetate (6), and p-hydroxybenzaldehyde (7). Compound 1 showed inhibitory activities against Staphylococcus epidermidis, Actinomyces viscosus, and Bacillus subtilis, with the minimal inhibitory concentrations of 62.5, 62.5, and 125μg/mL, respectively, while other compounds were inactive at the test concentrations. Compounds 1–7 demonstrated no inhibitory activities against a-glucosidase. [Conclusion] The paper reported the fungi from coral reef sand and their natural products for the first time, which enriched the knowledge about the diversity of fungi in coral reef sand and their natural products and laid a foundation for further research on such active natural products.

Abstract:[Background] Cellulose is a biomass resource to be developed and utilized, which is important for solving energy crisis and environmental pollution. [Objective] To isolate cellulase-producing bacteria from cow manure compost and investigate their cellulose degradation ability. [Methods] The cellulose solid plate Congo red staining method was used for preliminary screening, followed by liquid fermentation and cellulase activity measurement for secondary screening. [Results] A bacterium with high cellulase activity was isolated and identified as Bacillus amyloliquefaciens strain N5. Single-factor analysis experiments demonstrated that strain N5 exhibited good tolerance to pH, temperature, and salinity. Orthogonal optimization experiments revealed that the optimal conditions for cellulase production by strain N5 were an initial fermentation pH of 5.0, fermentation time of 96 hours, and fermentation temperature of 40 ℃. Under these conditions, the enzyme activity reached 189.27 U/mL. Furthermore, strain N5 achieved a 19.35% reduction in weight of rice straw within 7 days, indicating its effective promotion of rice straw degradation. Scanning electron microscopy results confirmed the ability of strain N5 to facilitate rice straw decomposition. [Conclusion] Strain N5 exhibits high cellulase activity and holds potential for developing efficient aerobic composting agents. It provides a valuable bacterial resource for the biological transformation of cellulose in solid waste.

Abstract:[Background] Erigeron breviscapus is a famous Chinese medicinal plant, while little is known about the diversity, community structure, and ecological roles of endophytic fungi (EF) in this plant. [Objective] To reveal the diversity, community structure, and ecological roles of EF in E. breviscapus. [Methods] The high-throughput sequencing of the ITS region was performed to profile the fungal community in the roots, stems, leaves, and flowers of E. breviscapus in Yunnan. FUNGuild was employed to predict the ecological functions of the fungi. [Results] A total of 540 operational taxonomic units (OTUs) were obtained from 12 samples. All of the OTUs were assigned into 188 genera, 114 families, 55 orders, 22 classes of 5 phyla. Only 14.45% of OTUs were shared by the four medicinal parts, and the unique OTUs were the most in the roots. Ascomycota and Basidiomycota were the dominant phyla in all the samples. The EF in the roots and flowers were dominated by Ascomycota and Basidiomycota, respectively. Didymella was the dominant genus, existing in all the four organs. Other dominant genera were Filobasidium, Cystofilobasidium and Plectosphaerella. The dominant and unique genera of EF varied in the four organs of E. breviscapus. The alpha diversity analysis showed that the roots had the highest richness of EF, while there was no significant difference in the diversity of EF among the four organs. Principal coordinate analysis indicated that the EF had similar community structures between leaves and stems, while the composition of EF in roots was different. FUNGuild prediction revealed that saprophytic fungi accounted for a high proportion in the EF of different samples and contained a large number of undefined taxa. [Conclusion] The EF of E. breviscapus has significant differences in community structure among different organs and demonstrates organ specificity. The findings provide a theoretical reference for the further development and utilization of EF resources in E. breviscapus.

Abstract:[Background] Plant seeds are important raw source for screening endophytic bacteria, from which the strains with great application values can be isolated. [Objective] To explore excellent endophyte resources from seeds, we identified the endophytic bacterial strain Fse32 isolated from the seeds of Dongxiang wild rice (Oryza rufipogon Griff.) and evaluated its resistance to pathogenic fungi and growth-promoting effects. [Methods] The isolated bacteria was identified based on morphological, physiological, and biochemical characteristics and 16S rRNA gene sequence. The antagonistic experiment was carried out to determine the inhibitory activities on pathogenic fungi. The tests of growth promoting ability, rice seed germination and pot experiments were conducted to evaluate the growth-promoting effect of the strain. [Results] Endophytic bacteria Fse32 was identified as Burkholderia gladioli Fse32. The strain exhibited remarkable effectiveness against Fusarium graminearum, Rhizoctonia solani, Sclerotinia sclerotiorum, Sclerotinia libertiana, Fusarium oxysporum, and Phytophthora capsici. Furthermore, Fse32 displayed excellen growth-promoting potential, the production of Indoleacetic acid (indole-3-acetic acid, IAA) at a yield of 17.95 mg/L, the capability of producing siderophores, with an A/A_r ratio of 0.24 and the inorganic phosphorus-solubilizing ability of 339.07 mg/L. The results of seed germination and pot experiments showed that strain Fse32 significantly promoted rice seed germination, and increased the plant height, root length, dry weight, and total chlorophyll content of rice seedlings by 7.9%, 33.5%, 108.9%, and 68.9% respectively. [Conclusion] Burkholderia gladioli Fse32 is an endophytic bacterium with diverse functions and high efficiency, demonstrating a promising prospect for biocontrol and growth promotion.

Abstract:[Background] Poa pratensis powdery mildew caused by Blumeria graminis f. sp. poae is an airborne disease, and planting resistant species is the most economical, environmentally friendly, and effective method for controlling this disease. [Objective] To observe the infection process of BGP(TG) on the leaves of three P. pratensis species with different resistance and clarify the role of papillae in the resistance of P. pratensis to powdery mildew. [Methods] Coomassie brilliant blue staining was combined with microscopy to observe the infection process of BGP(TG). The rate of secondary germ tube formation and the formation of haustoria and effective papillae in the three species were determined. [Results] The powdery mildew symptoms in ‘Explorer’ were more serious than those in the other two species, and the infection process of BGP(TG) was similar in different species. However, the formation of primary haustoria on the leaves of ‘Explorer’ was earlier than that of the other two species. One- or two-days post inoculation with BGP(TG), the formation of fifth-level germ tubes on the surface of ‘Taihang’ was slower than that of ‘Explorer’ and ‘Black Jack’. One day post inoculation with BGP(TG), more effective papilla was formed on ‘Taihang’ than on ‘Black Jack’, and the effective papilla formation rate on ‘Black Jack’ was higher than that on ‘Explorer’ (P＜0.05). [Conclusion] This study clarifies the infection process of B. graminis f. sp. poae and provides a theoretical basis for the prevention and control of powdery mildew in P. pratensis.

Abstract:[Background] The application of plant growth-promoting bacteria is an important measure for the sustainable development of agriculture. The seed-associated bacteria can interact with plants in the early lifecycle, being of great significance to plant growth. [Objective] To screen out the seed-associated bacterial strains with the function of promoting plant growth and inhibiting plant pathogens, to underpin the application of the strains in agricultural production. [Methods] The plant growth-promoting bacteria were isolated from the surface and the inside of peanut seeds. The roles of the isolates in promoting plant growth (nitrogen fixation, phosphorus solubilization, and IAA and siderophore production) and inhibiting the growth of plant pathogens were examined. The 16S rRNA gene sequencing was employed to determine the taxonomic status of the isolates. The biofilm formation and rhizosphere colonization tests were carried out to measure the viability of the strains in plant rhizosphere. Finally, pot experiments were conducted to examine the effects of the plant-growth promoting bacteria on the seed germination and seedling growth of peanut. [Results] A total of 41 plant-growth promoting bacterial strains were isolated from peanut seeds. All of the strains were capable of producing IAA, and 35, 2, and 14 strains showed the abilities of fixing nitrogen, secreting siderophores, and inhibiting plant pathogens, respectively. The representative strains (the surface/inside of peanut seed and the inside of peanut radicle), PS3, PE5, and PR5, were chosen according to the results of phylogenetic analysis and identified as Bacillus sp. The three strains formed folded biofilms on the surface of MSgg liquid medium and colonized the surface of peanut roots. They increased the seed germination rate and promoted the seedling growth of peanut. Specially, PS3, PE5, and PR5 increased the seed germination rate from 14.17% to 38.33%, 30.83%, and 39.17%, respectively, on day 2. Compared with the control, PS5 increased the seedling height, root length, fresh weight, and dry weight by 21.82%, 22.20%, 37.11%, and 35.64%, respectively; PE5 increased them by 17.45%, 18.93%, 26.10%, and 21.18%, respectively; PR5 increased them by 23.11%, 23.92%, 38.66%, and 37.47%, respectively. [Conclusion] The bacterial strains with high efficiency in promoting plant growth were isolated from peanut seeds, and significantly promoted seed germination and seedling growth. The strains provide potential resources for agricultural production, with excellent application potential.

Abstract:[Background] In the directed fruiting mode, the number of fruiting bodies produced affects the quality and yield of individual fruiting bodies due to the limitation of the fruiting area. However, the effects of artificial bud thinning on the quality and yield of Lentinula edodes remain to be studied. [Objective] To reveal the effects of artificial bud thinning (retaining different number of fruiting body buds) on the yield and quality of L. edodes in directed fruiting. [Methods] The substrate utilization in each group was monitored. The morphological indexes, yield, and texture characteristics of fruiting bodies after harvest were measured. [Results] The group with 10–20 mushroom buds retained showed better agronomic traits than other groups, with a single fruiting body weight of 15.74–23.76 g and a cap diameter of 45.98-52.37 mm. The group with 30 mushroom buds retained had the highest yield (368.9 g) of first-batch mushrooms. Artificial bud thinning had little effect on the texture of the stalk. With the increase in the number of mushroom buds retained, the hardness, gluing, reversibility, and chewiness of the mushroom caps decreased. The group with 10–20 mushroom buds retained had the best quality and the best texture. Moreover, this group showed high crude protein, crude fat, and amino acid content in the fruiting body. [Conclusion] Retaining 15–20 mushroom buds in each bag can help to obtain high yield and quality. The mushroom quality is the best in the group with 10 buds retained, which, however, decreases the biological efficiency and increases the workload. Therefore, we suggest retaining 15–20 buds in each bag.

Abstract:[Background] The grape production in eastern Helan Mountains has the problems of poor soil nutrients, neglect of organic fertilizer application, and environmental pollution caused by the burning of fruit plant branches. [Objective] In view of the problems caused by long-term application of chemical fertilizers to the soil, we carried out field experiments to study the effects of fertilizer application and fungicide spraying on the soil physicochemical properties and the fungal community composition and diversity, aiming to underpin the sustainable and healthy development of wine grapes. [Methods] The physicochemical properties of the rhizosphere soil of ‘Cabernet Sauvignon’ were examined. Illumina MiSeq high-throughput sequencing was performed to determine the fungal community composition and diversity in the rhizosphere soils in seven treatments: conventional fertilization (CK), earthworm manure+fermented branches+100×fungicide (T1), earthworm manure+fermented branches+ 200×fungicide (T2), earthworm manure+fermented branches+300×fungicide (T3), earthworm manure+unfermented branches+100×fungicide (A1), earthworm manure+unfermented branches+200×fungicide (A2), and earthworm manure+unfermented branches+300×fungicide (A3). [Results] Compared with CK, other treatments significantly changed the chemical properties of the rhizosphere soil. Specifically, the fertilization treatments increased the organic matter, did not alter the soil pH, improved the soil structure, and activated the available nutrients in soil. Compared with CK, The number of fungal operational taxonomic unit (OTU) decreased in all treatments, and A2 treatment increased the fungal richness and diversity in the rhizosphere soil. Ascomycota, Basidiomycota, Chytridiomycota, and Mortierellomycota were dominant in all the seven treatments at the phylum level, accounting for 74.40%–86.97% of the total relative abundance. T2 and A2 treatments increased the grape yield by 19.34% and 14.72%, respectively. The correlation analysis showed that total nitrogen was the main factor affecting the fungal community structure; microorganisms were not significantly related to yield; electric conductivity and total nitrogen were factors closely associated with yield. [Conclusion] T2 and A2 treatments improved the soil microbial community structure and soil nutrients, which promoted grape growth and improved yield and production efficiency, providing a theoretical basis for the selection of suitable fertilization schemes for grapes.

Abstract:[Background] Galium spurium, one of the main noxious weeds in crop fields, is mainly controlled by chemicals, which causes serious harm to the human body and the environment. Microbial herbicides with precise targets and environmental safety have practical significance for agricultural modernization. [Objective] To screen out the non-polluting and harmless strains with strong weed-controlling activities and provide new strain resources for the development of microbial herbicides. [Methods] The strains were isolated and purified by tissue culture method. The herbicidal activity and selectivity of the isolate were determined by the spore suspension method. Internal transcribed spacer (ITS) and EF-1/EF-2 gene sequencing was used to identify the isolate. The phylogenetic tree was constructed in MEGA 7.0 to reveal the phylogenetic relationship of the strain. Finally, the environmental conditions suitable for growth and sporulation of the strain were analyzed based on colony diameter and mycelial dry weight combined with blood cell counting. [Results] A strain DT-08C was isolated and identified as Fusarium paeoniae. The incidence rate of DT-08C infection in G. spurium reached 47.22%–93.93%, and the strain was safe to broad bean, pea, maize, Chinese cabbage, tomato, cucumber, and pepper. The optimum media for colony growth and sporulation of DT-08C was PSA and OA, respectively. [Conclusion] F. paeoniae DT-08C is highly pathogenic to G. spurium but safe to broad bean, pea, maize, Chinese cabbage, tomato, cucumber, and pepper, and it has wide adaptability to the environment. The results provide strain resources for the further field application.

Abstract:[Background] Phytophthora infestans is a major pathogen causing potato late blight. Stress-activated protein kinases (SAPKs), a class of mitogen-activated protein kinases (MAPKs), play a role in regulating cell responses of fungi to external stress factors. The biological functions of a SAPK in P. infestans, i.e., PiSAK1, remain unknown. [Objective] To explore the biological roles of PiSAK1 in the growth and development, the responses to external stress factors, and the infection in potato. [Methods] The bioinformatics tools were used to characterize PiSAK1. The expression levels of PiSAK1 at different development and infection stages were determined by RT-qPCR. Finally, the PiSAK1-silenced and-overexpressed strains were constructed and characterized. [Results] PiSAK1 had a typical Ser/Thr protein kinase catalytic domain of MAPKs and shared the same evolutionary branch with the SAPKs of other oomycetes. The expression level of PiSAK1 was the highest at the stage of cysts or 48 h after infecting potato. Moreover, it was significantly up-regulated after exposure to 0.3 mol/L NaCl or 3 mmol/L H₂O₂ for 0.5 h. The results showed that the PiSAK1-silenced strains were significantly weaker than the wild type in terms of colony growth, sporangium production, resistance to NaCl and H₂O₂, scavenging of reactive oxygen species, and pathogenicity. However, the PiSAK1-overexpressed strains showed significantly enhanced phenotypic characteristics compared with the wild type, except for no significant difference in colony growth. [Conclusion] PiSAK1 plays a role in the growth and development, osmotic regulation, oxidative stress response, and infection in potato. Our results provide a theoretical basis for further research on new targets of fungicides and breeding of resistant potato varieties.

Abstract:[Background] Pear ring rot caused by Botryosphaeria dothidea is one of the major diseases in pear cultivation, causing huge economic losses. [Objective] To identify the strain FJAT-55034 with high antagonistic activity against B. dothidea and evaluate its growth characteristics and antifungal activity, so as to provide an alternative strain for the biocontrol of pear ring rot. [Methods] Morphological characteristics, physiological and biochemical properties, and 16S rRNA gene sequencing were employed to identify the antagonistic strain FJAT-55034. Furthermore, we studied the growth characteristics of the strain by establishing the growth curve and determining the culture temperature and pH. The inhibition zone method was employed to determine the inhibitory spectrum of this strain. Finally, co-culture, microscopic observation, and pathogen inoculation in fruits were conducted to evaluate the antagonistic effect of strain FJAT-55034 on B. dothidea. [Results] The antagonistic strain FJAT-55034 was identified as Bacillus velezensis. It can survive at 20–50 ℃ (optimum 30 ℃) and pH 5.0–9.0 (optimum pH 7.0) and grow well with 0–5% NaCl. The strain exerted inhibitory effects on 6 pathogenic fungal species derived from fruit trees, with the inhibition zone diameters ranging from 19.8 mm to 29.1 mm. The inhibition rate of FJAT-55034 on the mycelial growth of B. dothidea was 77.2%. Moreover, the strain inhibited the expansion of pear ring rot with an inhibition rate of 66.0%. [Conclusion] The strain FJAT-55034 presented a desirable inhibitory effect on B. dothidea and served as an alternative strain for the biocontrol of pear ring rot.

Abstract:[Background] In view of the large area of Camellia oleifera with low efficiency, revealing the rhizosphere soil microorganisms affecting plant resistance and growth is essential for the sustainable development of forestry. [Objective] To understand the microbial community characteristics in the rhizosphere soils of native and introduced varieties of C. oleifera in Guangdong Province. [Methods] High-throughput sequencing was employed to analyze the microbial composition in the rhizosphere soil of C. oleifera. [Results] There were 676 species of bacteria belonging to 593 genera, 377 families, 201 orders, 77 classes, 26 phyla and 631 species of fungi belonging to 502 genera, 266 families, 121 orders, 50 classes, 14 phyla in the rhizosphere soil of C. oleifera. Acidobacteriota and Proteobacteria were the dominant bacteria and Ascomycota and Basidiomycota the dominant fungi. The rhizosphere soil microbial composition was significantly different between the native and induced varieties of C. oleifera. The bacterial diversity was significantly higher in the rhizosphere soil of the native variety than that of the introduced variety. At the phylum level, Desulfobacterota, Rozellomycota, and Mortierellomycota showed significantly different relative abundance between the two varieties. Amorphotheca was specifically enriched in the rhizosphere soil of the native variety. The relative abundance of carbon metabolism was significantly different between the rhizosphere soils of two varieties. Saprophytic fungi were dominant in the rhizosphere soil of C. oleifera, followed by pathotrophic and symbiotrophic fungi. Saprophytic fungi were significantly enriched in the rhizosphere soil of the native variety, while symbiotrophic fungi (especially arbuscular mycorrhizal fungi) had significantly lower relative abundance in the rhizosphere soil of the native variety than that of the introduced variety (6.43% vs. 21.83%). In addition, the organic matter and nutrients were key factors affecting the rhizosphere soil microbial community of C. oleifera. [Conclusion] The microbial community composition and structure in the rhizosphere soils of native and introduced C. oleifera were significantly different. Amorphotheca and arbuscular mycorrhizal fungi were significantly enriched in native and introduced C. oleifera, respectively. Nutrients may be the key factors affecting microbial communities in the rhizosphere soil of C. oleifera.

Abstract:[Background] Vinegar Daqu is an important source of microorganisms during the brewing of traditional grain vinegar in China. It is usually prepared once and used in batches. [Objective] To reveal the structural variations of the microbial community in the traditional vinegar Daqu during the long-term storage. [Methods] From a century-old vinegar factory in southern Shanxi, we collected the raw materials of Daqu and the vinegar Daqu products that were newly produced and had been stored for 7 and 12 months, respectively. Microbial diversity was investigated by high-throughput DNA sequencing. [Results] A total of 610 fungal operational taxonomic units (OTUs) and 747 bacterial OTUs were obtained from the four groups of samples. Ascomycota (95%) and Firmicutes (81%) were dominant among fungi and bacteria, respectively. About one third of fungal OTUs and 95% of bacterial OTUs in vinegar Daqu were detected in the raw materials of Daqu, indicating that raw materials acted as the main source of microorganisms in vinegar Daqu. Compared with that in the newly-produced vinegar Daqu, the diversity of fungi and bacteria decreased significantly in vinegar Daqu samples that have been stored for 7 months and 12 months. Microbial community structure changed during the storage of vinegar Daqu, with the bacterial community structure being more prone to fluctuation than the fungal community. Compared with those in the raw materials, fungi like Saccharomycopsis fibuligera and Issatchenkia orientalis and bacteria such as Kroppenstedtia were significantly enriched in vinegar Daqu products. According to the results of function prediction, the fungi were mainly saprotrophic, and the metabolism pathways of bacteria were enriched significantly. [Conclusion] Vinegar Daqu presented decreased microbial diversity and varied microbial community structure during the long-term storage. This study enriched our understanding of the microorganism source in traditional vinegar Daqu and the variation patterns of microbial community structure in vinegar Daqu during storage, providing a scientific basis for stabilizing Daqu quality and improving the production process.

Abstract:[Background] Riemerella anatipestifer (RA) is a Gram-negative bacterium that causes infectious serositis in ducks. Despite the extensive studies about the iron transport system of this bacterium, little is known about the function of iron-sulfur cluster assembly gene. [Objective] Sequence analysis shows that B739_RS03695 encodes an iron sulfur binding protein in R. anatipestifer CH-1, while the function of this gene remains unknown. This study aims to reveal the role of gene B739_RS03695 in the strain resistance to oxidative stress and antibiotics. [Methods] We constructed the B739_RS03695-deleted strain and studied its response to oxidative stress by establishing the growth curve and conducting the oxidative stress assay. The role of the gene in antibiotic resistance was measured by the minimum inhibitory concentration (MIC) and time-dependent killing experiment. [Results] Compared with the parent strain, RA CH-1ΔB739_RS03695 presented unaffected growth in the GCB medium, inhibited growth in the iron-restricted medium, increased sensitivity to H₂O₂, and enhanced resistance to gentamicin. The real-time PCR results showed that compared with the control group, the transcription level of B739_RS03695 was significantly up-regulated under iron restriction and hydrogen peroxide stress conditions. [Conclusion] The deletion of B739_RS03695 impaired the growth of RA CH-1 in iron-restricted medium. B739_RS03695 was required for the resistance of RA CH-1 to H₂O₂ and the killing effect of gentamicin on RA CH-1.

Abstract:[Background] Small colony variants (SCVs) play a role in the antibiotic resistance and persistent residues of bacteria, while little is known about the Escherichia coli (EC) SCVs from animals in China. [Objective] To study the biological characteristics of EC-SCVs from animals in Xinjiang Uygur Autonomous Region of China, so as to provide basic data for the related studies of EC-SCVs. [Methods] EC from the animals in Xinjiang Uygur Autonomous Region of China was induced with aminoglycosides to form SCVs. The cultural characteristics, biochemical properties, susceptibility to antibiotics, motility, biofilm formation, and hemolytic activities of the wild type and SCVs were examined. [Results] Five strains of EC-SCVs (2 heme-dependent and 3 unknown nutrient-dependent) were obtained from bovine, sheep, and horse by induction with kanamycin or gentamicin. EC-SCVs showed different biochemical characteristics from the wild type. All the EC-SCVs did not utilize acetate and showed varied cultural characteristics in different media. Moreover, they presented enhanced tolerance to aminoglycosides, increased biofilm formation, and decreased motility. Except that the hemolytic activity of heme-dependent EC-SCVs enhanced, the hemolytic activities of other SCVs changed little. [Conclusion] The biological properties of animal-derived EC-SCVs are different from those of the wild type, and these changes may pose great challenges to the prevention and control of the infectivity or drug resistance of E. coli. The related mechanisms remain to be studied in depth.

Abstract:[Background] OpaR is the master quorum sensing regulator of Vibrio parahaemolyticus. QsvR, an AraC-type transcriptional regulator, has reciprocal regulatory activities with OpaR. The regulatory effect of QsvR on gene expression is affected by OpaR, while their relationship in gene regulation remains to be fully elucidated. [Objective] To investigate QsvR regulons in the wild type (WT) and the opaR mutant (ΔopaR), and thus determine the effect of OpaR on the gene regulation of QsvR in V. parahaemolyticus. [Methods] Illumina HiSeq was employed to mine the differentially expressed genes in the qsvR mutant (ΔqsvR) or ΔqsvRΔopaR relative to that in WT or ΔopaR under the biofilm formation growth condition. [Results] QsvR regulated 1 735 genes (regulon 1) in the WT background, including 855 genes activated and 880 genes repressed. QsvR regulated 1 187 genes (regulon 2) in the ΔopaR background, including 533 genes activated and 654 genes repressed. There were 517 common genes shared by regulons 1 and regulons 2, and most of these genes were oppositely regulated by QsvR between the two regulons. According to the results of gene ontology (GO) annotation, 467 and 204 genes from regulons 1 and regulons 2 were respectively annotated to three categories (biological process, molecular function, and cellular component) and thirty sub-categories. The results of Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment showed that 372 and 678 genes from regulons 1 and regulons 2 were respectively enriched in 30 signaling pathways (Q value＜0.05). The genes in regulon 1 were mainly enriched in cellular metabolism, genetic information processing, and environmental information processing, and those in regulon 2 in cellular metabolism. The classification results obtained with the cluster of orthologous groups of proteins (COG) showed that the genes in regulons 1 and regulons 2 were mainly involved in amino acid transport and metabolism, signal transduction mechanisms, energy production and conversion, general function prediction only, and function unknown. In addition, the regulons 1 and regulons 2 contained a large number of regulatory genes and putative c-di-GMP metabolism-associated genes, as well as some polar flagellar genes, capsular polysaccharide genes, exopolysaccharide synthesis genes, type IV pili-associated genes, type III secretion system genes, and type VI secretion system genes. [Conclusion] QsvR was a global regulator in V. parahaemolyticus, controlling the transcription of a large number of genes. OpaR affected the regulatory actions of QsvR on its target genes and the composition of QsvR regulon.

Abstract:[Background] Biofilm formation is one of the major reasons for the increasing drug resistance of Pseudomonas aeruginosa, and the quorum sensing (QS) system plays a key role in biofilm formation. [Objective] QS inhibitors can inhibit the formation of biofilm and the secretion of virulence factors, serving as a new approach to address drug resistance. We chemically altered both the parent nucleus and acyl side chains of the las QS signaling molecule N-(3-oxododecanoyl)-l-homoserine lactone (OdDHL) to synthesize N-undecanoyl cyclopentamide, named Y0-C11-HSL, aiming to reveal the effects of Y0-C11-HSL on the biofilm formation and virulence factor secretion of P. aeruginosa and the underlying molecular mechanism. [Methods] Cystal violet staining and scanning electron microscopy (SEM) were employed to evaluate the effects of Y0-C11-HSL on the biofilm formation and structure. The inhibitory activity of Y0-C11-HSL was assessed by measuring the production and movement of virulence factors. The effect of Y0-C11-HSL on the surface chemical groups of extracellular polymers (EPS) was investigated by Fourier transform infrared spectrometry (FT-IR), and the mechanism of action of Y0-C11-HSL was studied by molecular docking. [Results] Compared with the control group, 10–200 μmol/L Y0-C11-HSL reduced the biofilm formation of P. aeruginosa, and the reduction rate reached 24.1% at 200μmol/L (P＜0.01). In addition, 200 μmol/L Y0-C11-HSL inhibited the secretion of pyocyanin, rhamnolipid, extracellular polysaccharide, and hydrolytic protease by P. aeruginosa, with the inhibition rates of 34.7% (P＜0.01), 33.1% (P＜0.01), 27.3% (P＜0.01), and 37.3% (P＜0.01), respectively. Furthermore, Y0-C11-HSL inhibited the swarming and twitching of P. aeruginosa, with the inhibition rates of 45.6% (P＜0.01) and 51.7% (P＜0.01), respectively, and it affected the EPS surface chemical groups. The molecular docking results showed that Y0-C11-HSL competitively bound to the OdDHL-bound LasR receptor. [Conclusion] Y0-C11-HSL could competitively bind to OdDHL-bound LasR receptor to affect the transcriptional proteins and thus down-regulate the expression of P. aeruginosa QS-related genes.

Abstract:[Background] The formation of dormant Mycobacterium tuberculosis (Mtb) is considered to be the main cause of latent tuberculosis infection (LTBI), the mechanism of which remains to be studied with in vivo and in vitro models. Mtb can infect mesenchymal stem cells (MSC) and survive in the cells in a dormant state for a long time. However, the modeling of Mtb-infected cells requires long time and high laboratory biosafety level. Therefore, it is essential to explore the available MSC for modeling Mtb infection and further deciphering the dormancy mechanism of Mtb. [Objective] To establish and characterize the human umbilical cord MSC (hUCMSC) model of Mycobacterium smegmatis (Ms) infection. [Methods] After hUCMSC were infected with Ms, flow cytometry was employed to identify the surface antigen. The cell membrane was labeled with DiI and then the phagocytosis of the hUCMSC infected with Ms was observed in a fluorescence microscope. The plate counting method was employed to measure the intracellular survival rate of Ms. The formation of lipid droplets in hUCMSC was observed by oil red O staining. Cytoskeletal changes were observed by fluorescence staining with phalloidin. The transcription levels of the genes involved in the lipid metabolism, resting, and differentiation of the hUCMSC infected with Ms were determined by RT-qPCR. [Results] Ms survived in hUCMSC in a non-replicating state, and the infection rate was positively correlated with the multiplicity of infection. Ms simulated the expression of the genes involved in lipid biosynthesis and inhibited the transcription of the genes involved in lipid degradation to promote the formation of lipid droplets in the Ms-infected hUCMSC. Ms promoted the cytoskeleton remodeling of hUCMSC. The RT-qPCR results showed up-regulated expression of the genes related to proliferation and differentiation, indicating that Ms promoted the differentiation of hUCMSC into lipoblasts and osteoblasts. [Conclusion] We successfully established a hUCMSC model of Ms infection, which could mimic the main characteristics of Mtb dormancy and be used in the study of LTBI mechanism.

Abstract:[Background] In recent years, 16S rRNA gene sequencing and metagenomic analysis have been commonly used for detecting microbial pathogens in the gut. [Objective] To overcome the limitations of high cost and long detection time, we established a method for analyzing the composition of human gut microbiota based on real-time fluorescence quantitative PCR (qPCR), which can measure the abundance of gut microorganisms. [Methods] Ten microbial taxa commonly detected in the intestine were selected from public databases. We designed specific primers and probes for the ten targets and validated them using 20 fecal samples. Furthermore, we compared the results of qPCR and 16S rRNA gene sequencing to evaluate the performance of the established method. [Results] The ten pairs of primers and probes were specific to the targeted taxa, and the coverage of targeted bacteria in the HITdb database exceeded 70%. The coefficient of variation (CV) of sample detection results was less than 10%, indicating that the method was highly stable. The results of qPCR and 16S rRNA gene sequencing for measuring the microbial abundance were correlated (P＜0.05). [Conclusion] The results of the relative abundance of microorganisms detected in fecal samples using the primers and probes designed based on the HITdb database were consistent with those obtained by 16S rRNA gene sequencing. This study provides an accurate and cost-effective method for analyzing gut microbiota and the reference information for clinical diagnosis and treatment.

Abstract:[Background] Gonorrhoea is one of the major sexually transmitted diseases in China, and the infection with Neisseria gonorrhoeae (NG) can promote the transmission and infection of HIV. The incidence of gonorrhoea in China is on the rise, and the emergence of multidrug resistant strains makes it an urgent need to develop protective vaccines for preventing the spread and infection of gonorrhea. [Objective] To unveil the advanced structure and epitopes of peptidyl-prolyl isomerase (PPIase) of NG, and explore the potential of PPIase as a vaccine and a molecular diagnostic target. [Methods] Bioinformatics tools were employed to analyze the polarity, hydrophilicity, flexibility, surface accessibility, secondary and tertiary structures, and T and B cell epitopes of PPIase. The prokaryotic expression system of PPIase was constructed with pET32a(+), and the recombinant protein was purified. The BALB/c mice were immunized with the purified recombinant protein and the NG cells disrupted by ultrasound, respectively, and the sera were then harvested. The surface antigens of NG whole cells were prepared. The binding of serum antibody induced by PPIase to the surface antigens of NG whole cells was examined by the enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence assay, respectively. [Results] The secondary structure of PPIase was mainly composed of α helix (39.34%), random coil (30.51%), and extended strand (20.59%). The epitope analysis revealed 5 B cell dominant epitopes, 9 cytotoxic T lymphocyte dominant epitopes, and 18 helper T cell dominant epitopes. The recombinant PPIase protein was expressed and purified by the prokaryotic expression system. The purified recombinant protein can stimulate BALB/c mice to produce high-titer antibodies, and the serum antibody induced by the recombinant PPIase protein can bind to the NG surface antigen. [Conclusion] PPIase as an outer membrane protein demonstrates good immunogenicity and immunoreactivity, and the specific antibody induced by this protein can bind to the surface antigen of NG. Therefore, PPIase has the potential as a candidate vaccine against NG.

Abstract:[Background] High-throughput screening technologies represented by flow cytometry can efficiently screen the microbial strains with target characteristics. In fluorescence-activated cell sorting (FACS), the adhesion of microorganisms will lead to inaccuracy results and low purity. Therefore, a rapid and simple method for preparing single cell suspension is crucial for FACS to obtain reliable results. Desired mutants are usually screened out in fluorescence-based manners from the libraries generated by random mutagenesis with low positive mutation rates. However, the impurities and dead cells with strong spontaneous fluorescence tend to be included into the sorting gate, which will lead to false-positive results and reduce the survival rates of samples. It is urgent to improve the survival rate of microorganisms in FACS. [Objective] To establish a simple method for preparing single cell suspensions for FACS and improve the survival rate of sorted microorganisms. [Methods] Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, and Saccharomycetes were used as the samples. The morphological characteristics and cell adhesion degrees of the live microorganisms were observed by microscopy. Four treatments, including ultrasound, digestive enzyme, surfactant, and ultrasound-surfactant, were then employed to alleviate cell adhesion. To improve the survival rate of microorganisms in FACS, we treated Saccharomyces cerevisiae HZ848 expressing green fluorescent protein by atmospheric and room temperature plasma (ARTP) to generate a mutant library. The top 0.5% of the whole cells and the negative cells after propidium iodide (PI) staining were separately sorted based on their GFP intensities, and the single-cell survival rates under both conditions were calculated. [Results] Yeast cells were effectively monodispersed by the treatment of 0.01% Tween-80 combined with ultrasound for 1 min, where the single cell rate was over 88%, and the breakage rate of PI-stained cells was lower than 1.4%. The single cell dispersion of C. glutamicum was achieved by the treatment with 0.01% Tween-80 combined with ultrasound for 5 min, where the single cell rate was over 97% and the breakage rate of PI-stained cells was lower than 1%. The survival rate of S. cerevisiae without PI staining was 4.3%, while this rate was raised to 18.3% for the PI-stained sample after removal of dead cells. [Conclusion] This study established a simple and rapid method to prepare single cell suspensions and provided a PI-staining strategy to significantly improve the survival rates of sorted samples in microbial FACS.

Abstract:[Background] Enterococcus faecium is one of ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens. Due to the resistance to a variety of antimicrobial agents, E. faecium seriously threatens human health and is included in the WHO priority pathogens list for R&D of new antibiotics. [Objective] To isolate, characterize, and sequence the genome of the virulent phage against E. faecium, so as to provide raw materials for the phage therapy of E. faecium. [Methods] A phage strain 1A11 against E. faecium was isolated from pasture sewage. The phage was observed in a transmission electron microscope, and its optimal multiplicity of infection, one-step growth curve, and lysis spectrum were determined. The biological characteristics of the strain were analyzed and the whole genome was sequenced. [Results] E. faecium phage 1A11 had a typical icosahedral head and a long tail, belonging to the Siphoviridae of the Caudovirales. The strain showed the optimum multiplicity of infection of 0.01, a lysis cycle of 70 min, an incubation period of 30 min, and an outbreak period of 40 min. It exerted specific lysis effects on several strains of E. faecium. Phage 1A11 had a genome of 42 750 bp with the GC content of 34.71%, 70 putative open reading frames (ORFs), and no antibiotic resistance genes or virulence genes. It can be used for the phage therapy of E. faecium. [Conclusion] A new strain of E. faecium phage 1A11 was isolated, which has certain research value and application potential, and laid a foundation for the study of E. faecium phage therapy.

Abstract:Lincomycin, an amide antibiotic produced by Streptomyces lincolnensis, is mainly used to treat infections caused by Gram-positive bacteria in clinical practice. In view of its high medicinal value and economic value, progress has been achieved in the biosynthesis and molecular regulation of lincomycin. This review introduces the structural features and biosynthesis of lincomycin, and summarizes the research progress in the molecular regulatory mechanism of lincomycin in S. lincolnensis. The review is conducive to the in-depth understanding of the secondary metabolic regulatory network of S. lincolnensis and provides theoretical guidance for the transformation of regulatory factors or their target elements in the strains with high yields of lincomycin.

Abstract:Among all digestive system tumors, pancreatic cancer is characterized by a high degree of malignancy and a low survival rate of patients. However, the specific mechanism of pancreatic cancer remains unclear. As the research on human microorganisms including intestinal microflora is flourishing in recent years, the potential pathogenesis of microorganisms about pancreatic cancer has become a hot topic of research. In this paper, we introduced the microbial markers associated with pancreatic cancer, analyzed their roles in pancreatic cancer, and expounded the effects of the tumor microenvironment formed by microorganisms on pancreatic cancer. This article provides a reference for the further study of the relationship between microorganisms and pancreatic cancer and facilitates the early examination, early prevention, and clinical treatment of pancreatic cancer by using microbiological technology in the future.

Abstract:Intestinal stem cells (ISCs) are the source of intestinal epithelial cells, maintaining intestinal homeostasis by orchestrating proliferation and differentiation. Gut microbiota and their metabolites also play a role in maintaining host intestinal homeostasis. With technological advances, the interaction between ISCs and gut microbiota has drawn increasing attention. Emerging evidence has demonstrated that ISCs regulate the composition of gut microbiota by affecting the subtypes of epithelial cells, while gut microbiota and their metabolites modulate the epithelial development mediated by ISCs. In this review, we elucidate the effects of ISCs differentiation on gut microbiota, summarize the progress in the effects of gut microbiota and their metabolites on the proliferation and differentiation of ISCs, and forecast the possible research directions in the future, aiming to give new insights into the therapies for intestinal injury.

Abstract:Biofilm is a special way for bacteria to survive. Antibiotic resistance, recurrence, and transfer of biofilm infection are urgent problems to be solved in clinical practice. Probiotics, as part of the microorganisms in the host, can restrain pathogens via a variety of ways. This paper summarizes the mechanisms of probiotics biofilm against pathogenic bacterial biofilm, and further reviews the measures for improving the research method and enhancing the stability of probiotics biofilms and developing new probiotics biofilms.

Abstract:Thraustochytrids, capable of producing multiple high-value natural active substances, such as eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), squalene, and carotenoids, have been recognized as an excellent source for commercial lipid production. Firstly, this paper briefed the ecological roles and biotechnological values of thraustochytrids and summarized two biosynthetic pathways of fatty acids. Secondly, we introduced the effects of NaCl, temperature, dissolved oxygen, and pH on the growth, lipid accumulation, fatty acid composition, and DHA production of thraustochytrids. Thirdly, we reviewed the research progress in the strategies for enhancing the DHA biosynthesis ability of thraustochytrids by osmotic regulation, staged fermentation, and alleviating oxidative stress based on environmental stress factors. Finally, we pointed out the existing problems in the current research on the molecular regulation mechanism, staged fermentation strategies, strain improvement, and metabolic engineering of thraustochytrids under environmental stresses, provided solutions for these problems, and prospected the possible development direction in the future. Overall, this review aimed to provide a practical reference for efficient industrial production of DHA by thraustochytrids.

Abstract:The intestinal virome is an important component of the human intestinal microecosystem. In recent years, the relationship between intestinal microbiome and diseases has attracted extensive attention. More and more evidence has demonstrated that the transfer of intestinal virome in fecal microbiota transplantation is essential for the efficacy of the therapy. By reviewing the relevant studies in recent years, this article summarizes the therapeutic potential and possible mechanisms of intestinal virome in fecal microbiota transplantation, and makes an outlook on the application of intestinal virome in disease treatment.

Abstract:According to the local industrial demands and the standards of national engineering education professional certification, our team implemented a project-oriented teaching reform of the course Environmental Microbiology and constructed an innovative teaching model that integrated teaching, learning, education, and application. We designed 3 teaching projects, 17 learning tasks, and 4 course objectives, improved the evaluation methods for achievements, and established a diversified assessment system. The reform was characterized by diverse teaching methods and forms and emphasized the unique teaching and ideological and political education. After theteaching reform, thestudents demonstrated improved initiative and enthusiasm in learning and achieved thegoals beyond expectation. The final student pass rate, comprehensive scores, supervision evaluation, peer evaluation, and student evaluation were all obvious improved. The project-oriented teaching reform of Environmental Microbiology achieved valuable results.

Abstract:“Environmental Engineering Microbiology” is a fundamental course in the field of environmental engineering. However, due to the abstract concepts and extensive content of Environmental Microbiology, most students have difficulty concentrating in class and struggle to understand and apply topics such as biological oxidation processes and the role of microorganisms in environmental engineering. As a result, the teaching of effectiveness is undesirable. In order to improve students’ learning efficiency and teachers’ teaching experience, a blended teaching reform and practice of “Environmental Engineering Microbiology” was carried out based on the Chaoxing Fan Ya online learning platform. The practical results show that the reformed course has stimulated students’ intrinsic motivation for learning, developed their ability for self-study and continuous learning, and achieved positive effects in terms of knowledge, skills, and overall qualities. It has effectively enhanced students’ professional abilities and comprehensive qualities. At the same time, the teaching methods, teaching abilities, teaching experience, and information technology of the course teachers have been improved and enhanced, strengthening the practice of course innovation and improving the teaching quality of Environmental Engineering courses.

Abstract:[Background] In recent years, the gut microbiology of insects has received extensive attention from researchers, and a large number of research papers and works have been published. However, the systematic bibliometric analysis of insect gut microbiota remains to be carried out. [Objective] To understand the history, hotspots, and trends of the research on insect gut microbiota at home and abroad. [Methods] We searched the China National Knowledge Infrastructure (CNKI) and Web of Science (WOS) with insect gut microbiota as the topic, and analyzed the keywords using the bibliometric tools VOSviewer and CiteSpace. [Results] The global research publications in this field showed an increasing trend during 1991–2022, and the research focuses and directions gradually expanded. The keyword clustering anchored three emerging research areas: insect meal, secondary metabolites, and biodegradation. [Conclusion] Bibliometric studies provide a rapid and intuitive understanding of the foundations, frontiers, and trends of the research on insect gut microbiota.

Abstract:[Background] Phosphorus-transforming microorganisms play an important role in the phosphorus cycle in the natural environment. These microorganisms promote the efficient resource utilization, reduce environmental pollution, and have positive impacts on food security and ecosystem stability by dissolving, mineralizing, absorbing, and transporting phosphorus in the environment. [Objective] To explore the research hotspots and trends of phosphorus- transforming microorganisms both domestically and internationally in recent years, visualize the knowledge structure and evolution process in this field, and provide feasible references and insights for subsequent research. [Methods] We retrieved the articles in this field published by China National Knowledge Infrastructure (CNKI) and Web of Science core collection (WOS) from 2002 to 2022. CiteSpace was employed to analyze the keyword co-occurrence, keyword clustering, bursts, number of publications, country distribution, author cooperation, and institution collaboration. [Results] A total of 887 valid articles were ultimately screened out. The research on phosphorus-transforming microorganisms began to flourish after 2016 and experienced accelerated growth in recent years. China and the United States were leaders in this field. China was the country with the largest number of publications, and the Chinese Academy of Sciences was the institution with the largest number of publications. The research hotspots included the classification and screening of strains, metabolic pathways, nutrient cycling, environmental pollution, and ecological protection. [Conclusion] Phosphorus-transforming microorganisms have broad development prospects. The mechanism of phosphorus- transforming microorganisms in response to biochar is becoming a new research hotspot. The wide application of such technologies in the future is an inevitable development trend. This article provides a visual explanation of the research and development trend of phosphorus-transforming microorganisms, proposes future key research directions, and provides a theoretical basis for agricultural production and sustainable development.