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  • QIAO Jiahuizi, SHEN Shuo, HU Rong
    Microbiology China. 2022,49(7):2411-2427
    DOI: 10.13344/j.microbiol.china.210902
    [Background] Bacillus atrophaeus E20303 can inhibit the activity of the dry rot pathogen of potato and the response surface methodology can optimize the components of medium for fermentation and fermentation conditions of biocontrol bacteria. The optimal medium components and fermentation conditions for E20303 can serve as a reference for the preparation and application of biocontrol agents against potato dry rot. [Objective] To optimize the medium components and fermentation conditions of E20303, a strain isolated from the mud of Chaerhan Salt Lake in Qinghai with high activity against the dry rot pathogen of potato, by response surface methodology in order to improve its antifungal activity. [Methods] Single factor experiment, central composite design, and response surface methodology were used to design and optimize the medium components and fermentation conditions of E20303. [Results] The optimal components of the medium were starch 10.72 g/L, yeast powder 23.60 g/L, MgSO4·7H2O 16.00 g/L, CaCO3 1.14 g/L, KH2PO4 8.00 g/L, and K2HPO4 16.00 g/L. After optimization, the antifungal rate increased from 46.51% to 62.00%. The optimal fermentation conditions were liquid medium volume of 102.89 mL, pH 8.64, 28.73 ℃, rotation speed 200 r/min, culture for 3 days, and inoculum 2%. Under the conditions, the bacteriostatic rate rose from 51.15% to 72.51%. [Conclusion] The fermentation formula and fermentation conditions of E20303 with significantly improved activity against the dry rot pathogen of potato were clarified, which laid a foundation for research on the biocontrol agents against potato dry rot.
    Citation
    QIAO Jiahuizi, SHEN Shuo, HU Rong.Improving the activity of Bacillus atrophaeus E20303 against the dry rot pathogen of potato: based on response surface methodology[J].Microbiology China,2022,49(7):2411~2427
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  • FENG Xinqian, LIU Shuqi, HANA xiangGUAN, Wanchun
    Microbiology China. 2022,49(7):2428-2441
    DOI: 10.13344/j.microbiol.china.211105
    [Background] Extreme weather events, such as typhoons, bring transient and lasting effects on aquatic ecosystems due to strong winds and precipitation. However, very few studies have focused on the effects of typhoons on aquatic microbial communities and antibiotic resistance genes (ARGs). [Objective] To understand the effect of extreme weather on freshwater ecosystem, we analyzed the microbial communities and ARGs in urban freshwater before and after typhoon. [Methods] Water samples were collected from four sites before and after the typhoon. The effects of Lekima on the microbial communities and ARGs in recreational waters in Wenzhou were examined by metagenomic analysis in summer, 2019. In addition to the aquatic microbial communities and ARGs, the physical and chemical parameters of water samples from each sampling site were analyzed, including temperature, pH, dissolved oxygen, chlorophyll a, soluble active phosphorus, nitrate, nitrite, andammonium. [Results] The pH, dissolved oxygen, and chlorophyll a at most sites increased after the typhoon, and the typhoon had a weaker effect on Jiushan Lake than on Sanyang Wetland. The relative abundances of Proteobacteria, Cyanobacteria, and Bacteroidetes increased, while that of Actinobacteria decreased after the typhoon. At the genus level, Limnohabitans showcased significantly increased diversity and relative abundance after the typhoon. Among all the environmental factors, ammonium was the key environmental factor affecting microbial community structure. In addition, 35 opportunistic pathogen taxa were detected in all samples. The relative abundance of Pseudomonas aeruginosa, an important opportunistic pathogen, increased after the typhoon. The ARGs showed spatial (among sampling sites) and temporal (before and after the typhoon) variation. A redundancy analysis showed that water total inorganic nitrogen was the main environmental factor affecting the distribution of ARGs.[Conclusion] These findings provide new insights into how extreme weather (e.g., typhoons) influences microbial communities and ARGs in freshwater system. Typhoon landing increases the public safety risk of urban freshwater system. Therefore, the testing and quarantine should be carried out in advance to strengthen the evaluation and analysis of environmental health and safety, which will help to reduce the risk of antibiotic resistance and pathogen diffusion.
    Citation
    FENG Xinqian, LIU Shuqi, HANA xiangGUAN, Wanchun.Effects of typhoon Lekima on microbial communities and antibiotic resistance genes in urban freshwater systems[J].Microbiology China,2022,49(7):2428~2441
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  • [Background] Municipal waste leachate is a kind of complex organic wastewater with high nitrogen content, which can causes serious environmental pollution if discharged directly into the environment. [Objective] To select heterotrophic nitrifying and aerobic denitrifying (HN-AD) strains that can tolerate high concentration of ammonia nitrogen in waste leachate and efficiently remove nitrogen from wastewater, and thereby provide functional strains to address nitrogen pollution in urban wastewater. [Methods] The strains that could tolerate high concentration of ammonia nitrogen were isolated from the waste leachate, and a strain with the strongest denitrification capacity was screened out and named U1. Based on 16S rRNA gene sequencing and physiological and biochemical characteristics, U1 was identified as Pseudomonas aeruginosa. Then we further investigated nitrogen removal ability of U1 in the presence of ammonia nitrogen of different initial concentration and different carbon sources, and at different rotation speeds, initial pH, and carbon-to-nitrogen ratios, and explored the optimal nitrogen removal conditions of U1 in combination with L9(34) orthogonal test. [Results] U1 with strong denitrification ability was isolated and the optimum denitrification conditions for this strain are as follows: ammonia nitrogen at initial concentration of 1 000 mg/L, mixed carbon source of brown sugar+ trisodium citrate, pH of 6.0, carbon-to-nitrogen ratio of 10, and rotation speed of 130 r/min. Under the optimum conditions, the maximum total nitrogen removal rate of U1 was 64.37% and the maximum ammonia nitrogen removal rate was 76.73%. For the waste leachate with total nitrogen and ammonia nitrogen amount of 2 345 mg/L and 1 473.8 mg/L, respectively, the maximum removal rate of total nitrogen by U1 was 27.86% and the maximum removal rate of ammonia nitrogen was 21.32%. [Conclusion] U1 can be used to treat wastewater with high-concentration ammonia nitrogen.
    Citation
    GAO Yajuan, JIN Jingchen, GAO Jie, GAO Yuxuan, WU Huijuan, ZHANG Wentian, LI Chenchen, ZHANG Guowei, JIN Yongsheng.Identification and denitrification characteristics of heterotrophic nitrification and aerobic denitrification strain U1 resistant to high concentrations of ammonia nitrogen[J].Microbiology China,2022,49(7):2442~2456
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  • [Background] Microorganisms play an indicative role in the environment, and the composition of microbial community is one of the research hotspots in water environment. [Objective] To explore the bacterial community structure and spatial distribution in the sediment of the Third Drainage Ditch in Ningxia. [Methods] The physicochemical properties of the sediment samples were analyzed, and the 16S rRNA gene amplifiers of bacteria in surface sediments from 11 sampling sites in gullies and main tributaries were sequenced by high-throughput sequencing technology. [Results] The sediment of the Third Drainage Ditch had weak alkalinity and spatial differences in physicochemical properties including organic carbon, total nitrogen, total phosphorus, ammonium nitrogen and nitrate nitrogen. The concentrations of organic carbon, total nitrogen, and total phosphorus in the sediment of Pingluo section were lower than those in Helan and Huinong sections. The richness and diversity of bacteria in the sediment of Huinong section (in the lower reaches) were significantly higher than those in Helan section (in the upper reaches). The dominant phyla were Proteobacteria (24.41%–44.40%), Chloroflexi (5.46%–17.55%), Actinobacteria (9.12%–21.21%), Bacteroidetes (6.96%–13.10%), Thermodesulfobacteria (3.40%–12.20%), Firmicutes (3.31%–14.61%), and Acidobacteria (2.00%–9.77%). The dominant genera were Thiobacillus (6.73%) and norank_f__Steroidobacteraceae (4.28%). The bacterial community composition in the sediment of Shi’er tributary was much different from that of the Third Drainage Ditch, while the composition was similar among the sampling sites in Huinong section. The redundancy analysis of bacterial community and environmental factors showed that total nitrogen, ammonium nitrogen and nitrate nitrogen had great influence on bacterial community structure in the sediment. [Conclusion] The bacterial community in the sediment of the Third Drainage Ditch showed obvious spatial differences and was greatly affected by nitrogen. The microorganisms involved in nitrogen cycle remain to be studied.
    Citation
    YANG Guiqin, CHANG Jie, ZHENG Lanxiang, GAO Li, LI Fujuan.Microbial community structure and spatial distribution in sediment of the Third Drainage Ditch in Ningxia[J].Microbiology China,2022,49(7):2457~2469
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  • [Background] Heavy metal pollution in Longjiang River has been a great concern since the “dam collapse” in 2012. [Objective] To reveal the bacterial community structure in nearshore sediments of heavy metal-polluted rivers and the environmental influencing factors. [Methods] With the high-throughput sequencing method (Illumina MiSeq PE300), we investigated the bacterial community characteristics in nearshore sediments of Longjiang River and the correlation with environmental factors. [Results] The average content of As, Cd, and Zn in the nearshore sediments of Longjiang River was 25.06, 3.20 and 205.36 mg/kg, 1.22, 11.97 and 2.72 folds of the soil environmental background values of Guangxi, respectively. The C/N ratio ranged from 3.66 to 13.15, with the average of 10.55, indicating that the organic nitrogen was mineralized in the sediments. The dominant bacterial phyla were Chloroflexi (12.16%–35.36%), Proteobacteria (7.69%–30.85%), Acidobacteria(8.56%–22.48%), Bacteroidetes (5.26%–30.41%), Desulfurobacteria (1.14%–10.65%), and Actinobacteria (1.55%–4.17%), and the dominant classes were Anaerolineae (8.92%–30.04%), Gammaproteobacteria (5.10%–24.98%), Bacteroidetes (1.54%–26.95%), Acidobacteria (1.69%–9.40%), Vicinamibacteria (1.23%–9.09%), and Alphaproteobacteria (2.42%–6.67%). Correlation analysis showed that the abundance of bacterial community in sediments was significantly correlated with NH4+-N and pH, and ACE and Chao 1 indexes of bacterial community were negatively correlated with NH4+-N (P<0.05). [Conclusion] The abundance and diversity of bacterial community in sediments were mainly affected by NH4+-N and pH, and the dominant communities were related to the biogeochemical cycle of carbon, nitrogen, phosphorus and sulfur. The sediments might be the home to some pollution indicator microorganisms and pathogenic microorganisms.
    Citation
    YAO Shengxun, MENG Jiangquan, LU Sufen, HUANG Changxiao.Characteristics of bacterial community structure in the nearshore sediments of Longjiang River and the environmental impact factors[J].Microbiology China,2022,49(7):2470~2485
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  • [Background] Skatole is the main component of organic pollutants in livestock compost, which can cause the deterioration of farms and surrounding environment. Therefore, skatole pollution needs to be solved urgently. [Objective] This study aims to isolate, identify and characterize an efficient skatole-degrading bacterial strain to provide an efficient strain for the degradation of skatole and lay a foundation for the application of this strain to the remediation of odor-polluted environments. [Methods] Using the minimal salt medium with skatole as the only carbon source, we isolated an efficient skatole-degrading strain from a pig manure compost sample. Strain YKSW-6 was identified based on morphological characteristics and 16S rRNA gene sequence, and its growth pattern and skatole degradation characteristics were analyzed. Gas chromatography-mass spectrometry (GC-MS) was employed to analyze the intermediates of skatole degradation. [Results] Strain YKSW-6 was identified as Rhodococcus gordoniae. When the strain was inoculated at a ratio of 10%, the degradation rate for 100 mg/L skatole reached 100% within 14 h. This strain can utilize 18 carbon sources such as D-sorbitol and bromosuccinic acid, and was resistant to 13 chemicals such as potassium tellurite and potassium bromate. YKSW-6 completely degraded skatole in the culture medium at 5% inoculum ratio, 30–42 ℃, and pH 6.0–9.0. The strain showed the best growth and skatole degradation performance at pH 7.2, 37 ℃ and 180 r/min. The GC-MS results showed that skatole was firstly oxidized at C2 to 3-methyloxindole, and then oxidized to N-(2-acetylphenyl)formamide by strain YKSW-6. Phenylacetaldehyde and phenylacetic acid were also the intermediates during the degradation. [Conclusion] Strain YKSW-6 is a Rhodococcus strain with strong skatole-degrading ability among the reported strains, which enriches the resource of skatole-degrading strains. The results provide a theoretical basis for the development and application of this strain.
    Citation
    ZHANG Zongyuan, ZHOU Liuzhu, YUE Dandan, GUO Wenyang, PAN Mengshi, QI Landa, WANG Xueyan, XU Hongguang, CHEN Guocan, ZHANG Yingtao.Isolation, identification and characterization of a skatole-degrading bacterial strain YKSW-6[J].Microbiology China,2022,49(7):2486~2499
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  • [Background] Anthropogenic disturbance affects urban wetlands and natural wetlands to different degrees. [Objective] To study the differences of microbial diversity in sediments of two different types of wetlands. [Methods] The sediment samples of Longfeng wetland and Dangnai wetland were collected in winter and summer. The bacterial and archaeal community structures were determined via high-throughput sequencing of 16S rRNA genes. The differences of bacteria and archaea between the two wetlands and the correlations between environmental factors and microorganisms were analyzed. [Results] The abundance of Thiobacillus, Bacillus, and Sphingomonas in the sediment of Longfeng wetland was higher than that of Dangnai wetland (P<0.05). The abundance of Methanoregula in Dangnai wetland was higher than that in Longfeng wetland. Anaerolinea and Methanosarcina had higher abundance in the sediment of Dangnai wetland than in Longfeng wetland in winter (P<0.05). [Conclusion] The differences between Longfeng wetland and Dangnai wetland mainly affect the abundance of methanogenic archaea and bacteria involved in element cycling in the sediment. Human disturbance and low temperature can reduce the diversity of microorganisms in the wetlands, and pH, salinity, and alkaline phosphatase are the main environmental factors that significantly affect the microbial diversity.
    Citation
    TIAN Runmin, ZHAO Duo, HONG Yanhua, ZHANG Hongqi, SHEN Guinan, YAN Lei, QIU Huajiao, WANG Weidong.Diversity of bacteria and archaea in sediments of Longfeng wetland and Dangnai wetland and its response to environmental factors[J].Microbiology China,2022,49(7):2500~2514
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  • [Background] Many protective bricks of the M2 mausoleum of Dingtao King have been unearthed, and some have suffered from fungal deterioration. [Objective] In this study, samples from the surfaces of five bricks were collected to examine and analyze the fungi in them. In the meanwhile, fungistatic experiment was carried out on the isolated seven fungi. [Methods] We use scanning electron microscopy, high-throughput sequencing, pure culture, and fungistatic experiment to examine and analyze the fungi in samples. [Results] The result suggested serious fungal deterioration on the surfaces of artifact bricks without fungicide treatment and alleviation of the deterioration on the surfaces after treatment with 75% ethanol and 0.5% miconazole nitrate. Fungal species were different depending on the bricks. To be specific, Boeremia was most abundant on brick 9 and Cordycipitaceae dominated brick 13. It was found that miconazole nitrate and the mixture of boracic acid and borax failed to inhibit the fungi, while the fungistatic product K100 (2-methyl-4-isothiazolin-3-one) was effective on them. [Conclusion] This study is of great significance for the analysis and control of fungi on the surfaces of bricks and for protection of bricks of artifacts.
    Citation
    WANG Ling, WANG Yu, YANG Xinyu, WANG Cen, LI Chenghao, CUI Shengkuan, PAN Jiao.Analysis and prevention of fungal deterioration on the surface and in the storage environment of bricks unearthed from the mausoleum of Dingtao King[J].Microbiology China,2022,49(7):2515~2526
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  • [Background] 16S rRNA gene amplicon sequencing, a culture-independent approach, is commonly used to identify the bacterial community structure and relative abundance in given samples. The protocol of high-throughput sequencing technology involves many experimental steps, and the nuances of each step may be magnified in the final sequencing results, resulting in unexpected deviations from the actual situation. [Objective] With MiSeq system, we evaluated the impact of five factors in PCR on 16S rRNA gene amplicon sequencing results: primer sequence, annealing temperature, template quantity, cycle number, and denaturation time. [Methods] The 16S rRNA gene amplicon of mock community DNA was sequenced to determine the effects of these five factors on the accuracy of the results achieved. [Results] Primer had great influence on the 16S rRNA sequencing results, and primer B (V3–V4, 341F/806R) showed the highest quantification accuracy among all primers, followed by primer A (V3–V4, 341F/805R). Among the different annealing temperatures (52, 55 and 60 ℃), the result of annealing at 60 ℃ was closest to the theoretical value. As for the DNA template quantity (2, 10 and 50 ng), the deviation was minimal when 2 ng DNA template was used. The result of detection with (20+8) cycles was closest to the theoretical value of mock DNA compared with that of the other three groups (15+18, 25+8, and 30+8). Denaturation time (3 min and 5 min) had no significant effect on the experimental results. [Conclusion] This study revealed that primer sequence, annealing temperature, template quantity, and number of cycles were important factors affecting the accuracy of 16S rRNA gene sequencing results, which laid a foundation for the selection of standardized methods of library construction.
    Citation
    ZHAO Feng, SHI Ya, WANG Ying, LIANG Qian, CHEN Huan, LI Yinghong, DOU Xiaobing, HE Luping.Effect of PCR conditions on the accuracy of 16S rRNA gene amplicon sequencing[J].Microbiology China,2022,49(7):2527~2537
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  • WANG Yuting, XU Tong, HUA Beijie, JIANG Juquan
    Microbiology China. 2022,49(7):2538-2549
    DOI: 10.13344/j.microbiol.china.211067
    [Background] Owing to the key roles of Zn2+ in cell detoxification and physiological processes, Zn2+ transporters have attracted increasing attention. In Escherichia coli, zntA and zitB are two key genes responsible for Zn2+ efflux. [Objective] This study aims to construct a Zn2+ sensitive mutant of E. coli and verify its function. [Methods] Using E. coli DH5α as the original strain, we knocked out zntA via a λ Red recombination system by using the homologous recombinant fragment carrying a kanamycin resistance gene. On the basis of the single-gene knockout mutant, zitB was further knocked out via the homologous recombinant fragment carrying a gentamicin resistance gene. In this way, a mutant with the knockout of both zntA and zitB was obtained and designated as KZAB04. The functional complementation experiment was conducted to test the sensitivity of the mutants and the original strain (negative control) under different Zn2+ concentrations. [Results] KZAB04 exhibited higher Zn2+ sensitivity than the original strain. [Conclusion] A Zn2+ sensitive mutant of E. coli was successfully constructed. The construction of this mutant is the prerequisite for the functional study of zntA and zitB and lays a foundation for the functional identification of other Zn2+ transporter genes.
    Citation
    WANG Yuting, XU Tong, HUA Beijie, JIANG Juquan.Construction and functional verification of a Zn2+ sensitive mutant of Escherichia coli[J].Microbiology China,2022,49(7):2538~2549
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  • [Background] Hydroxynaphthalene reductase (HNR), a key enzyme in the biosynthesis of 1,8-dihydroxynaphthalene (DHN) melanin, is involved in fungal melanin synthesis and has regulatory effect on the fungal growth and pathogenicity. However, the regulatory role of HNR in the infection structure differentiation of fungal pathogens remains unknown. [Objective] On the basis of the cloning and bioinformatics analysis of the hnr genes of Alternaria alternata, we preliminarily evaluated the regulatory role of HNR in the growth and infection structure differentiation of A. alternata through pharmacological methods, aiming to provide a theoretical basis for revealing the molecular mechanism of HNR regulating the infection structure differentiation of A. alternata. [Methods] The hnr genes were cloned by homologous cloning method, and the nucleotide and amino acid sequences were analyzed via bioinformatics tools including gene structure display server, open reading frame Finder, conserved domain search. The HNR-specific inhibitor tricyclazole was used for the study about the regulatory role of hnr in the growth and development, melanin synthesis, and infection structure formation of A. alternata. Real-time quantitative PCR (RT-qPCR) was employed to determine the expression levels of hnr genes at the spore germination (2 h), appressorium formation (6 h), and hypha formation (8 h) stages throughout the infection structure differentiation of A. alternata. [Results] The full-length coding sequences (CDSs) of two hnr genes were cloned from A. alternata and designated as Aa4hnr and Aa3hnr. Aa4hnr had a length of 1 266 bp, encoded 268 amino acid residues, and contained 9 ORFs and no intron. Aa3hnr, with a length of 1 356 bp, encoded 267 residues and contained 2 introns (51 bp and 49 bp) and 17 ORFs. The phylogenetic analysis showed that Aa4hnr and Aa3hnr shared high homology with the hnrs from Ophiobolus disseminans and Alternaria arborescens, respectively. The presence of NAD(P) binding domain indicated that Aa4hnr and Aa3hnr belonged to the short-chain dehydrogenase/reductase (SDR) family. Tricyclazole treatment significantly reduced the DHN melanin synthesis in A. alternata and inhibited the infection structure formation of A. alternata on hydrophobic surface. The expression of Aa4hnr was down-regulated at all the stages of infection structure differentiation, and that of Aa3hnr was down-regulated at the appressorium formation stage (6 h) while significantly up-regulated at the hypha formation stage (8 h). [Conclusion] Aa4hnr and Aa3hnr have some regulatory effects on infection of A. alternata.
    Citation
    LI Rong, XU Wenyi, LI Yongcai, BI Yang, ZHANG Miao, JIANG Qianqian, LIU Yongxiang.Cloning of hnr and characterization of its regulatory role in the infection structure differentiation of Alternaria alternata[J].Microbiology China,2022,49(7):2550~2562
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  • [Background] Fruiting body is the important structure for fungal reproduction and growth and its development is regulated by a variety of signaling pathways. [Objective] In this study, we analyzed the genes in pheromone signaling pathway in Flammulina filiformis on the basis of the transcriptome and genome data to obtain differentially expressed genes, and then determined their expression during mycelia growth and fruiting body development, hoping to provide a reference for studying the fruiting body development of edible fungi. [Methods] We annotated the pheromone signaling pathway of F. filiformis based on genomic data. The key genes involved in the development of F. filiformis fruiting body in this pathway were further identified through transcriptome sequencing. Finally, we verified the key genes by real-time fluorescent quantitative PCR. [Results] The expression of cdc24 and ste12 gene was significantly different in the five samples (primordium, stipe at the elongation stage, pileus at the elongation stage, stipe at the mature stage, and pileus at the mature stage) collected during fruiting body development. PCR results supported the above conclusion. [Conclusion] The key genes cdc24 and ste12 may be involved in the regulation of tissue differentiation during fruiting body development of F. filiformis.
    Citation
    LIU Ao, LYU Xiaomeng, WANG Qingji, WANG Xiangfeng, LI Zhuang, ZHANG Jiyue, WANG Wei.Differential expression of key genes in pheromone signaling pathway in fruiting body development of Flammulina filiformis[J].Microbiology China,2022,49(7):2563~2574
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  • [Background] Naked barley root rot caused by pathogenic fungus such as Fusarium avenaceum and Bipolaris sorokiniana are widely occurred, which poses a great threat to agricultural production and development in Tibetan areas. However, there is still a few research on its biocontrol by bacterial inoculant in China. [Objective] To screen strains with excellent disease-controlling and plant growth-promoting properties from the healthy naked barley rhizosphere. Furthermore, we explored the interaction effects among the bacteria to screen the excellent inoculant formulas, and the bacterial inoculants were made accordingly. After all, our ambition is to provide superior microbial inoculants which catering for the biocontrol of naked barley root rot, promoting the crop yield as well. [Methods] Firstly, we used the selective media to screen growth-promoting strains and plate confrontation method to screen the disease-controlling strains. After determining the pathogenicity of the bacteria by root irrigation and foliar inoculation, the nitrogen fixation capacities, phosphorus dissolution capacities, and IAA secretion capacities of them were determined by acetylene reduction method, molybdenum blue colorimetric method and high performance liquid chromatography, respectively. Additionally, the bacteria were identified by 16S rRNA gene sequence analysis. Finally, the biocontrol effects of the inoculants were measured by field test and evaluated by TOPSIS comprehensive analysis method. [Results] A total of 23 strains with nitrogen-fixing and phosphorus-solubilizing abilities were isolated in the initial screening, and 6 bacteria among them with eminent antagonistic ability, including 5 organophosphorus solubilizing strains, 3 inorganic phosphorus solubilizing strains and 3 nitrogen-fixing strains, all of which secreted IAA. Besides, the pathogenicity of the bacteria showed no pathogenicity to common crops in the sampling area, such as naked barley, oat, rapeseed and pea. According to the identification result, the bacteria isolates include 2 Bacillus subtilis, 1 Bacillus pumilus, 1 Bacillus cereus, 1 Pseudomonas fluorescens and 1 Acinetobacter sp. As a result, we firstly optimized 7 combinations (SC1−SC7) and then selected the best 4 (SC2, SC4, SC6, SC7) as inoculant formulas. Moreover, they were all made into liquid, powder and accumulative bacterial inoculant severally. Eventually, the field plot biocontrol determination showed SC7 (Bacillus pumilus Xpq-3, Bacillus cereus Xpq-15 and Acinetobacter sp. Xpq-21) had the best biocontrol and plant growth-promoting effects. [Conclusion] It’s clear that there are amount of biocontrol and growth-promoting strains in the naked barley rhizosphere soil. In addition, the microbial inoculant made by them have excellent biocontrol effects.
    Citation
    XU Shiyang, LI Minquan, LIU Meijin, XU Dongli, QI Yonghong, ZHOU Lanlan, WANG Xuemiao, LI Fengqing, LI Xueping.Screening of bacteria with biocontrol effects against naked barley root rot accompany with plant growth-promoting properties and the biocontrol effects of the bacterial inoculant[J].Microbiology China,2022,49(7):2575~2586
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  • [Background] Colletotrichum gloeosporioides, featuring a wide range of hosts and diverse infection methods, causes serious agricultural disasters. The biological functions of CgGcp1 in C. gloeosporioides, a C2H2 transcription factor, have not been reported. [Objective] This study aims to clarify the biological functions of CgGcp1, which is expected to lay a theoretical basis for clarifying the pathogenic mechanism of the fungus. [Methods] The CgGCP1-knockout vector was constructed and the knockout mutant was yielded by homologous recombination. The biological functions of CgGCP1 were identified by pathogenicity analysis and phenotypic analysis, such as the vegetative growth, stress response, conidium production, and appressorium formation. [Results] The growth rate of the CgGCP1-knockout mutant was lower than that of the wild type, and the mutant was more sensitive to SDS, Congo red, NaCl, and glycerol. The conidium yield, appressorium formation rate, and invasion ability were obviously decreased, and its virulence to rubber leaves was also lowered significantly. [Conclusion] CgGcp1 regulates the vegetative growth, cell wall integrity, conidium production, appressorium formation and invasion, and pathogenicity of C. gloeosporioides.
    Citation
    ZHANG Xingyuan, WANG Diguang, GAO Jing, TANG Wen, LI Xiaoyu.Biological functions of a C2H2 transcription factor CgGcp1 in Colletotrichum gloeosporioides[J].Microbiology China,2022,49(7):2587~2598
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  • WU Tianshang, XUE Yarong, LIU Changhong
    Microbiology China. 2022,49(7):2599-2611
    DOI: 10.13344/j.microbiol.china.211034
    [Background] Biocontrol of plant diseases and development of biocontrol products have been the research focuses in plant protection. However, most of the existing biocontrol agents feature a few available dosage forms (wettable powder or aqueous solution), short shelf life, and inconvenient transport and use. [Objective] To optimize the fermentation medium of Bacillus velezensis CC0955 and develop a novel biocontrol agent “effervescent granules”, which is easy to use and preserve. [Methods] Plackett-Burman design, central composite design, and response surface methodology were used to optimize the composition of CC0955 fermentation medium. With the evaluation indicators of pH and bacterial viable count, the composition of CC0955 effervescent granules was optimized by L9(33) orthogonal design, and the physical properties and antifungal effect of the effervescent granules were assessed. [Results] The optimal fermentation medium for CC0955 was composed of peptone 12.00 g, yeast powder 1.00 g, glucose 15.00 g, MgSO4·7H2O 0.40 g, K2HPO4 0.05 g, and distilled water 1 L. After 48 h fermentation with this medium, the inhibition of the culture broth on Rhizoctonia solani reached 89.78%. The optimal composition of effervescent granules is as follows: alkali-to-acid molar ratio of 2.00, silica of 1.50 g, and potassium fulvic acid of 0.03 g. The effervescent granules showed the average disintegration time of 61 s and the pH of the disintegrated solution was 5.44. In addition, the half maximal effective concentration (EC50) against R. solani was 27.65 mg/mL, and the number of viable bacteria was 6.05×108 CFU/g. The survival rate of bacteria was 58.68% after six months of storage at room temperature. Pot experiment showed that the control rate of CC0955 effervescent granule solution on rice sheath blight was 42.35%, and that on tomato gray mold was 57.62%. [Conclusion] The CC0955 effervescent granule is a novel formulation against phytopathogens, which can be used for the control of farmland diseases.
    Citation
    WU Tianshang, XUE Yarong, LIU Changhong.Optimization of fermentation medium and preparation of effervescent granules of Bacillus velezensis CC0955[J].Microbiology China,2022,49(7):2599~2611
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  • [Background] As common biocontrol bacteria, Bacillus exhibit great potential in the prevention and control of plant diseases. [Objective] In this study, we optimized the fermentation conditions and evaluated the stability of Bacillus kochii DDWB with nematicidal activity, aiming to provide theoretical support for the development and application of the strain. [Methods] With the nematicidal activity of supernatant and the OD600 of fermentation broth as indexes, we optimized the culture medium and fermentation parameters of DDWB strain via single factor tests and orthogonal design. Furthermore, we analyzed nematicidal activity about pH, temperature, UV irradiation, heredity, and storage stability of the fermentation broth. [Results] The optimized culture medium for DDWB strain contained 2% sucrose, 1% yeast extract, and 2% potassium chloride. The optimized fermentation conditions were initial pH 8.0, the filling volume of 150 mL/250 mL conical flask), the fermentation time of 48 h, the inoculum volume of 8%, the rotation speed of 160 r/min, and the fermentation temperature of 31 ℃. The fermentation broth was sensitive to acid and alkali, and its nematicidal activity degraded after 4 h of UV irradiation. However, the fermentation broth was not sensitive to temperature, and the genes related to nematicidal activity could be stably inherited. [Conclusion] We optimized the fermentation conditions and evaluated the stability of active substances in the fermentation broth of DDWB strain, aiming to make the strain rapidly propagate and maintain its long-term and stable nematicidal activity against root-node nematodes. The findings laid a foundation for evaluating the field efficacy and biocontrol mechanism of DDWB strain.
    Citation
    XU Yufei, ZHANG Xiaomin, ZHU Jiamei, WANG Qingbin, ZHANG Xiaoying, WANG Hongfeng, LIU Feng, MU Wei.Optimization of fermentation conditions and evaluation of stability of Bacillus kochii[J].Microbiology China,2022,49(7):2612~2624
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  • [Background] Endophytic bacteria play an important role in crop growth, development and stress tolerance. In this study, an endogenous bacterial strain Pseudomonas moraviensis GF-55 was isolated from maize. [Objective] To analyze the role of GF-55 in the growth and lodging resistance of maize. [Methods] Pot trial was conducted to analyze the role of GF-55 in the growth of maize seedlings and greenhouse trial to study the lodging resistance of maize after GF-55 inoculation. [Results] The pot trial results showed that GF-55 treatment increased the height, dry weight, and fresh weight of maize seedlings by 43.47%, 26.67%, and 82.44%, respectively, compared with the control. Furthermore, it increased the dry weight, fresh weight, length, volume, surface area, and mean diameter of maize root by 231.25%, 96.42%, 141.68%, 46.51%, 37.07%, and 52.38%, respectively. The strain had the indole-3-acetic acid (IAA) production, siderophore relative content, and phosphorus-solubilizing ability of 30.88 μg/mL, 50.20%, and 58.43 mg/L, respectively. The greenhouse trial demonstrated that GF-55 treatment significantly improved the stalk resistance of maize after the silking stage. Specifically, GF-55 treatment increased the stalk puncture, bending, and crushing strength of maize by 15.78%, 55.83%, and 33.71%, respectively, compared with the control. After GF-55 inoculation, the hemicellulose, cellulose, and lignin content in the stalk increased by 10.56%, 2.91%, and 48.01%, respectively. [Conclusion] P. moraviensis GF-55 promoted the growth of maize at the seedling stage and enhanced the lodging resistance after silking stage. It has important application value in maize growth promotion and lodging resistance and provides theoretical support for the development and utilization of this strain.
    Citation
    BAI Jianfei, HAN Shengcai, GAO Julin, YU Xiaofang, QING Ge'er, HU Shuping, ZHANG Sainan, GUO Jiang'an.Endophytic bacterial strain GF-55 improves the growth and lodging resistance of maize[J].Microbiology China,2022,49(7):2625~2637
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  • [Background] Infection with Clostridium perfringens causes necrotizing enteritis in animals, which is commonly treated by antibiotics. With the limitation of antibiotics in feed and husbandry in China, probiotics and its metabolites have become the focus of current research. [Objective] This study was conducted to reveal the properties of B. subtilis BS-2 against Clostridium perfringens. [Methods] The growth curve of B. subtilis BS-2, the antimicrobial properties of its metabolites and the mRNA levels of bacteriocin gene cluster were detected. [Results] The metabolites of B. subtilis BS-2 had no inhibitory effect on Gram-negative bacteria, whereas they had strong activity against Gram-positive bacteria. The activity of the strain against C. perfringens increased rapidly within 2–12 h and remained stable within 12–24 h. The antibacterial properties of the metabolites were not affected by pepsin, trypsin or protease K, which demonstrated good thermal stability. The mRNA levels of bacteriocin gene cluster showed that the inhibitory activity of B. subtilis BS-2 against C. perfringens was related to the expression of surfactin and mersacidin. [Conclusion] B. subtilis BS-2 may inhibit C. perfringens by regulating the expression of surfactin and mersacidin.
    Citation
    HUANG Jinxiu, CHEN Li, SU Guoqi, GUAN Xiaofeng, WANG Ruisheng.Characteristics of Bacillus subtilis BS-2 against Clostridium perfringens[J].Microbiology China,2022,49(7):2638~2647
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  • [Background] Continuous cropping causes imbalance in microbial community structure, deterioration of soil environment, poor nutrient cycling, and thus the yield reduction of Angelica sinensis (Oliv.) Diels. Therefore, it is imperative to improve soil by modern microbial technology and reduce continuous cropping obstacles. [Objective] To explore the effects of complex bacterium inoculants on rhizosphere soil enzyme activities, available nutrients, and yield of A. sinensis, clarify the yield-improving mechanism, and improve the measures for increasing yield. [Methods] The phosphorus-solubilizing activity of different strains was detected based on the halo zone, nitrogen-fixing activity by acetylene reduction assay, and peroxidase activity and nitrifying ability with kits. Complex bacterium inoculants T1 (Pseudomonas fluorescens CBS5, P. alcaligenes CBS7, P. extremaustralis CBSB, Zoogloea ramigera CBS4), T2 (P. fluorescens CBS5, P. alcaligenes CBS7, P. extremaustralis CBSB), and CK (sterile potato dextrose broth) were used to treat A. sinensis. The activities of enzymes related to nutrient cycling and transformation, and the content of available nutrients such as nitrogen, phosphorus and potassium in rhizosphere soil were determined by spectrophotometry. The yield was measured with conventional method. Statistical software was employed for variance analysis and principal component analysis of data. [Results] CBS7 had poor phosphorus-solubilizing ability. All the strains had nitrogen-fixing ability, peroxidase activity, and nitrifying ability, which were particularly prominent in CBS4. Compared with the CK, T1 and T2 significantly increased the activities of enzymes related to soil carbon and nitrogen metabolism and decreased the activity of soil alkaline phosphatase at the vigorous growth stage and late growth stage of A. sinensis root. They improved the content of available potassium, nitrate nitrogen, ammonium nitrogen, and available nitrogen in soil, content of ammonium nitrogen and nitrate nitrogen in root, and the nitrate reductase activity in root. Moreover, they promoted the root growth of A. sinensis and increased the yield. Compared with T2, T1 significantly enhanced the activities of sucrase and urease in soil, and raised the content of ammonium nitrogen and available nitrogen in soil and the content of ammonium nitrogen in root during the vigorous growth stage of root, and T1 improved the content of nitrate nitrogen and available nitrogen in soil, content of ammonium nitrogen and nitrate nitrogen in root, and nitrate reductase activity in root at the late growth stage of root. [Conclusion] T1 and T2 activated potassium in rhizosphere soil and improved the proportion of nitrate nitrogen in rhizosphere soil by nitrification to alleviate the toxicity of the high-concentration ammonium salt to root. The ratio of nitrate nitrogen to ammonia nitrogen in root was increased by active transport of nitrate nitrogen in A. sinensis. Then the nitrate reductase activity in root was improved, promoting the transformation of nitrate nitrogen to ammonium nitrogen and nitrogen utilization. In a word, T1 and T2 promoted the absorption and utilization of nutrients, particularly T1.
    Citation
    YANG Tao, YAO Yangyang, WANG Zhiye, DANG Shengrong, MAO Ting, NIU Yongyan, PENG Tong, WANG Yinquan.Effects of complex bacterium inoculants on rhizosphere soil nutrients and yield of Angelica sinensis in continuous croppin[J].Microbiology China,2022,49(7):2648~2660
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  • [Background] With the enhancement of drug resistance, the extended spectrum beta-lactamase-producing Klebsiella pneumoniae (ESBL-KP) seriously threatens food safety and human health. Blueberry, rich in polyphenols and anthocyanins, is a potential natural antibacterial candidate. [Objective] To analyze the inhibitory activity and mechanism of blueberry extracts against three ESBL-KP strains and test the effect of food matrix (cow milk) on the antibacterial activity of the extracts. [Methods] Fruit extracts of Vaccinium corymbosum L. and V. ashei were prepared with methanol, and the content of total phenols and anthocyanins in the extracts was determined. The antibiotic resistance of K. pneumoniae was detected by disk diffusion test. The antibacterial activity of blueberry extracts was evaluated based on minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and growth curves of strains. The effect of blueberry extracts on cell membrane integrity was detected by confocal laser scanning microscopy, and its antibacterial activity in milk was detected. [Results] The total phenol content in fruit extracts of V. ashei and V. corymbosum L. was 2.3 mg/g and 3.5 mg/g, respectively, and the anthocyanin content was 67.5 mg/100 g and 92.5 mg/100 g respectively. The MIC and MBC of two blueberry extracts against K. pneumoniae KP106, KP305, and KP408 were 25 mg/mL and 50 mg/mL, respectively. The growth curve showed that the two extracts at 2xMIC could kill K. pneumoniae within 6–8 h. Confocal laser scanning microscopy demonstrated that blueberry extracts could destroy the integrity of cell membrane. The simulated cow milk sample test showed that 100 mg/mL fruit extract of V. corymbosum L. could completely inhibit the growth of ESBL-KP in milk. [Conclusion] Blueberry extract exerts inhibitory effect on K. pneumoniae by destroying the cell membrane of the bacteria, and has the potential to prevent and control K. pneumoniae in milk. This study provides a theoretical reference for the application of blueberry in the prevention and control of drug-resistant K. pneumoniae in milk.
    Citation
    LIU Danyang, CHEN Changrong, LAN Weiqing, ZHAO Yong, SUN Xiaohong.Inhibitory activity of blueberry extract against extended spectrum β-lactamase-producing Klebsiella pneumoniae[J].Microbiology China,2022,49(7):2661~2672
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  • YANG Huimin, REN Xiaona, QIN Yi
    Microbiology China. 2022,49(7):2673-2685
    DOI: 10.13344/j.microbiol.china.211082
    [Background] Ganzi, an alpine wine region in southwestern China, enjoys favorable natural conditions for making wine. [Objective] To study the diversity of eukaryotic microorganisms, the genetic diversity of native Saccharomyces cerevisiae, and the diversity of native yeasts under the influence of commercial yeasts in the Ganzi wine region. [Methods] Cabernet Sauvignon grapes were grouped for Inoculated fermentation and natural fermentation. Then, Internal Transcribed Spacer (ITS) high-throughput sequencing was used to analyze the diversity of microorganisms during fermentation, and Interdelta fingerprinting was employed for classifying the genotypes of wild S. cerevisiae identified by 26S rRNA gene. [Results] Yeasts in 7 genera of 7 families in the natural fermentation and Inoculated fermentation were respectively identified, and Interdelta fingerprinting showed 5 genotypes of S. cerevisiae in Ganzi wine region. According to the phylogenetic tree of 6 representative S. cerevisiae strains in the Ganzi wine region and 109 S. cerevisiae strains from other producing areas in China, the 6 strains were in close genetic relationship with those from Beijing. [Conclusion] Ganzi wine region is rich in yeast resources, showing high microbial diversity and moderate genotypic diversity of native S. cerevisiae, which presents a potential for searching excellent native strains.
    Citation
    YANG Huimin, REN Xiaona, QIN Yi.Diversity analysis of yeasts in the Ganzi alpine wine region, Sichuan[J].Microbiology China,2022,49(7):2673~2685
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  • [Background] Exopolysaccharide (EPS) is a secondary metabolite produced during the growth and metabolism of lactic acid bacteria. In addition to improving the texture and quality of products, its physiological function has also attracted the interest of scholars in recent years. [Objective] This study intends to explore the characterization and molecular structure of EPS of lactic acid bacteria, and to reveal the relationship between it and the probiotic properties of EPS. [Methods] The EPS produced by Streptococcus thermophilus MGB80-7 was studied. To be specific, the yield of EPS was determined with the phenol-sulfuric acid method. Then the EPS was separated and purified by ion exchange column chromatography and gel molecular sieve chromatography, and the phenotypic structure of EPS was analyzed based on gel chromatography, infrared spectroscopy, and high performance liquid chromatography. As for the antioxidant activity, the ability to scavenge superoxide anion, hydroxyl radical, and DPPH radical was detected. [Results] The EPS yield of MGB80-7 in M17 medium was high, which was (268.25±5.36) mg/mL. After separation and purification, two components were obtained. Among them, the neutral polysaccharide (WPS-807, 1.028×105 Da) was composed of glucose, galactose, and mannose, and a small amount of rhamnose and arabinose, and the acidic polysaccharide (SPS-807, 8.601×104 Da) was made up of complex monosaccharides, which was dominated by mannose, galactose, and arabinose. EPS-807 can scavenge some free radicals, especially the hydroxyl radicals. [Conclusion] The molecular weight and structure of EPS produced by MGB80-7 influence its antioxidant activity. This result lays a basis for further analysis of the structure-activity relationship and antioxidant mechanism of EPS from lactic acid bacteria.
    Citation
    QIAO Shaoting, XIE Min, Daiannaer, HU Haimin, HE Binbin, DAN Tong.Phenotypic structure and antioxidant activity of exopolysaccharide produced by Streptococcus thermophilus MGB80-7[J].Microbiology China,2022,49(7):2686~2699
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  • [Background] Brucellosis threatens human health and the development of animal husbandry and vaccines with low virulence, good immunogenicity, and no interference of serological diagnosis turn to be a solution. [Objective] To compare the dynamic changes of specific T cells and antibodies in mice immunized with rough-type RA343 and smooth-type A19 of Brucella, and to evaluate the immune effect of rough-type RA343. [Methods] The 6-week-old female BALB/c mice were classified into three groups, 15 in RA343 group, 15 in A19 group, and 5 in blank control group. For RA343 group and A19 group, each mouse was injected (sc) with 0.1 mL (1×108 CFU) bacterial solution through into groin. A total of 5 mice in each of the three groups were killed 1, 2, and 3 weeks after immunization, respectively, and their spleens were separated. A part of the spleen was ground, and lymphocytes were separated to detect the proportions of CD4+T cells and CD8+T cells in mice (flow cytometry). The other part was weighed and then RA343 and A19 of Brucella were isolated, to evaluate the colonization of RA343 and A19 in mouse spleen. At the same time, the peripheral blood was collected and the serum was separated. For the determination of IgM and IgG in the serum of immunized mice (ELISA). GraphPad Prism 8.0 was employed for plotting and statistical analysis. [Results] The proportion of Brucella-specific CD4+IFN-γ in spleens (P<0.01), and the proportions of CD4+IL-2, CD8+IL-2, and CD8+TNF-α (P<0.05) increased in RA343 group compared with those in A19 group 1 week after immunization. Two weeks after immunization, the proportion of specific CD4+TNF-α (P<0.01) and the proportion of CD8+TNF-α (P<0.05) in RA343 group rose compared with those in A19 group. Three weeks after immunization, RA343 group registered increase of the proportion of CD4+TNF-α (P<0.05) and the proportion of CD8+IFN-γ (P<0.01) compared with A19 group. The bacterial content in spleens of RA343 group was lower than that of A19 group (P<0.05), and the spleen weight was also lower than that of A19 group 1–3 weeks after immunization. IgM and IgG levels of mice in A19 group increased significantly 2 weeks after immunization and no IgM or IgG was detected in the RA343 group. [Conclusion] Compared with A19, RA343 induced strong cellular immune response. The bacterial content in spleen of mice in RA343 group was significantly lower than that in A19 group and no serological cross-reaction occurred between antibody and A19 antigen. In summary, RA343 has low virulence, and can induce strong cellular immune response and humoral immune response in mice, with no interfere of serological diagnosis. Thus, it can be used as a candidate strain for the development of vaccine against brucellosis.
    Citation
    YANG Xiaotong, ZHANG Yinghui, LI Qiaoling, JIANG Hui, DING Jiabo, SHEN Qingchun, FAN Xuezheng, QIN Jianhua.Dynamic changes of specific T cells and antibodies in mice immunized with rough-type RA343 and smooth-type A19 of Brucella[J].Microbiology China,2022,49(7):2700~2714
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  • [Background] Collagenase can efficiently degrade water-insoluble collagen and is widely used in various industries. Most of the collagen from animal remains in fishing grounds of freshwater lakes will settle into sediments, and thus the ecological environment should be rich in collagenase-producing strains. [Objective] The collagenase-producing strains were screened from the sediments of freshwater lakes around Dongting Lake. Among them, a strain with high enzyme production and biosafety was identified and the fermentation conditions for enzyme production were optimized. The result is expected to lay a foundation for the industrial production and application of collagen and collagenase. [Methods] The collagenase-producing strains were screened with the spread plate method and the spot inoculation method. The collagenase activity of fermentation broth was determined by the ninhydrin colorimetry. Enzyme-producing strains were identified by 16S rRNA gene sequence alignment and phylogenetic analysis. Antibiotic sensitivity and haemolytic activity were detected with the disk diffussion method, amino acid decarboxylase activity by the bromocresol purple chromogenic assay, and nitrate reductase activity by nitrate reductase activity assay kit. Single factor experiment and orthogonal experiment were carried out to optimize the fermentation conditions for producing collagenase. [Results] A total of 113 collagenase-producing strains were screened out. Among them, a strain with high enzyme production and biosafety was selected from the sediment of East Lake in Huarong County, which was identified an Exiguobacterium strain and named Exiguobacterium sp. DJ1. The optimal enzyme production conditions of strain DJ1 were as follows: in 100 mL medium of 30.0 g/L gelatin, 10.0 g/L peptone, 5.0 g/L fish meal, 0.5 g/L KH2PO4, 0.2 g/L MgSO4·7H2O, 0.1 g/L NaCl in a 250 mL flask, with the initial pH of 8.5, inoculum of 3%, and culture temperature of 28 ℃. Under the optimized conditions, the activity of collagenase reached (185.45±23.87) U/mL, which was about 3.7 times higher than that before optimization. [Conclusion] There are abundant collagenase-producing strains in the sediments in fishing grounds of freshwater lakes. Strain DJ1 has high enzyme-producing capacity and biosafety. After optimization of fermentation conditions, the enzyme activity of strain DJ1 is increased by about 3.7 times, which lays a theoretical foundation for the industrial application of collagen and collagenase.
    Citation
    TANG Bailu, TAN Duoduo, LI Mengjiao, LI Jingni, ZHOU Zhijie, WANG Zihan, CHEN Zhongyuan, XIA Hu, HE Hailun.Screening of a collagenase-producing strain from the sediments in fishing grounds of freshwater lakes and optimization of the fermentation conditions[J].Microbiology China,2022,49(7):2715~2729
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  • [background] The Gram-negative Methylobacterium has the ability to grow in the low-carbon compound (such as methane, methanol, formaldehyde and so on) substrate with one carbon atom or no C−C bond, and synthesizes various metabolites such as amino acids, industrial enzymes, cofactors, polyhydroxyalkanoates (PHAs), polysaccharides, and carotenoids. [Objective] In this study, mutation of mutS gene in Methylobacterium sp. MB200 was performed, and targeted induction was conducted with stresses to yield mutant strains that can tolerate high concentration of methanol or formaldehyde. [Methods] The mutS deletion strain MB200sTB was constructed by triparental mating, and the concentration of methanol and formaldehyde in the culture medium was gradually increased to induce targeted mutations. Electrotransformation was used to yield complementary transformants. [Results] MB200sTB with mutS deletion was successfully constructed, and the methanol-tolerant MB200sHBc and formaldehyde-tolerant MB200sHBq were obtained. The methanol tolerance of MB200sHBc was greatly improved compared with that of the wild strain MB200. To be specific, the tolerant methanol concentration raised from 8 g/L to 44 g/L and the growth kept unchanged. In the presence of 0.45g/L formaldehyde as carbon source, the growth of MB200sHBq was 1.69 folds higher than that of MB200. [Conclusion] Through targeted induction of mutS deletion mutants, strains with high methanol tolerance and potential application prospect in production can be obtained.
    Citation
    YANG Qingshan, HUANG Luodong, WU Yiting, ZHANG Xi, SHEN Peihong.Mutation of mutS gene in Methylobacterium sp. MB200 and induction of strains resistant to high concentration of methanol and formaldehyde[J].Microbiology China,2022,49(7):2730~2740
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  • [Background] Escherichia coli O157:H7 is one of the most important foodborne pathogens. The accelerated drug resistance of E. coli O157:H7 invokes the selection of phages with strong lytic properties to achieve the purpose of disease prevention and treatment with broad application prospects. [Objective] To isolate and identify lytic phages against E. coli O157:H7 and analyze their biological characteristics and bacteria-killing efficiency, which may provide insights into the research and application of phages. [Methods] Phages were isolated and purified using double-layer plate method, and their morphology was observed by transmission electron microscope (TEM). The optimal multiplicity of infection (MOI), one-step growth curve, pH and temperature stability were determined, and the whole genome of phages was sequenced. Lysis efficiency was determined by a single phage or cocktail method in contaminated beef. [Results] Two strains of lytic E. coli O157:H7 phages FEC14 and FEC19 were isolated and purified. According to TEM results, the heads of the two phages were both icosahedral, and FEC14 presented a head diameter of about 80 nm and star-shaped tail filaments, while FEC19 had a head diameter of about 58 nm and needle-shaped tail filaments. Moreover, FEC14 showed an optimal MOI of 0.001, the latent period of 15 min and the lysis period of 65 min with an average burst size of 156 PFU/cell, and FEC19 displayed an optimal MOI of 0.1, the latent period of 10 min and the lysis period of 80 min with an average burst size of 800 PFU/cell. FEC14 stabilized at 60 ℃ and pH 4.0–11.0, while FEC19 survived at 70 ℃ and pH 5.0–9.0. FEC14 belongs to the genus Kuttervirus, family Ackermannviridae, order Caudovirales, and FEC19 falls into the genus Wifcevirus, family Myoviridae, and order Caudovirales. The single phage or phage cocktail effectively inhibited the growth of host bacteria in the contaminated beef, and the two phages had no known virulence genes and drug resistance genes, showing great potential efficacy in the control of E. coli O157:H7 contamination. [Conclusion] Two strains of lytic E. coli O157:H7 phages FEC14 and FEC19 with high specificity, stability and lysis efficiency reveal their potential to be an alternative antimicrobial agent for food safety in the future.
    Citation
    LI Zhaoxue, LAN Guanda, FAN Congcong, XU Cuiting, GONG Yunwei, CUI Wei, ZHAO Chunyan.Characterization of enterohaemorrhagic Escherichia coli O157:H7 phages FEC14 and FEC19 and their potential use in contaminated beef[J].Microbiology China,2022,49(7):2741~2752
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  • [Background] Alkaline protease, a fermentation product of many Bacillus species, is an important enzyme in industry. [Objective] An alkaline protease-producing strain was isolated by casein medium from an environmental sample. The passage number, carbon source, nitrogen source, metal ions, phosphate, initial pH, inoculum amount and temperature of the fermentation were optimized to increase the alkaline protease production of the strain and to reduce the cost of fermentation. [Methods] The isolates were identified via Gram staining, scanning electron microscopy, physiological and biochemical tests, and 16S rRNA sequence analysis. The fermentation conditions were optimized by single factor test, Plackett-Burman experiment, the steepest ascent method, and response surface methodology. The experimental data were analyzed in Minitab. [Results] The strain was identified as B. licheniformis and named B. licheniformis NWMCC0046. The optimized formula of the fermentation medium was determined as soybean meal 50.00 g/L, glucose 10.00 g/L, yeast extract 13.46 g/L, CaCl2 0.50 g/L, Na2HPO4·12H2O 4.00 g/L, and KH2PO4 0.30 g/L. The fermentation conditions were optimized as pH 7.5, 34.81 ℃ and the inoculum amount of 4.13%. Under these conditions, the alkaline protease activity reached 165.41 U/mL in 48 h of shake flask fermentation, which was 8.04 times higher than that before optimization. [Conclusion] The optimization of the medium formula and fermentation conditions improved the ability of B. licheniformis to produce alkaline protease, providing a reference for subsequent research on alkaline protease.
    Citation
    ZHOU Wei, ZENG Songyu, YU Jinfeng, XIANG Jun, MA Zhongren, DING Gongtao, ZHOU Xueyan, ZHANG Fumei.Optimization of alkaline protease production by a strain of Bacillus licheniformis[J].Microbiology China,2022,49(7):2753~2766
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  • FANG Huiying, ZHANG Gong
    Microbiology China. 2022,49(7):2767-2777
    DOI: 10.13344/j.microbiol.china.211025
    Due to the extensive use of antibiotics, the drug resistance of bacteria has become prominent, threatening human health and economy. The genetic and molecular mechanisms of bacterial drug resistance have been intensively investigated, while the corresponding regulatory mechanism has been rarely explored. Translation regulation, one of the most important regulation methods in organisms, has been underestimated in the research on bacterial resistance. This review introduced the main mechanisms of antibiotics affecting the translation process, and systematically summarized the research progress of bacterial resistance based on translation regulation from the aspects of ribosomal modification and mutation, dynamic regulation of total tRNA, tRNA aminoacylation, tRNA methylation, ribosomal protective proteins and translation factors, etc. We hope to provide a new perspective on the bacterial drug resistance, as well as some new ideas for the development of novel translation-targeted antibiotics.
    Citation
    FANG Huiying, ZHANG Gong.Drug resistance of bacteria based on translation regulation: a review[J].Microbiology China,2022,49(7):2767~2777
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  • ZHU Chenyang, WU Xinyuan, XUE Yongchang
    Microbiology China. 2022,49(7):2778-2788
    DOI: 10.13344/j.microbiol.china.211030
    Peptidyl carrier protein (PCP) is a core module in non-ribosomal peptide synthetase (NRPS). According to the assembly mechanism, each module of NRPS contains at least one PCP domain. PCP plays a role in the delivery of amino acid residues and polypeptides in different catalytic domains, and it serves as a platform to transfer amino acid residues and polypeptides to other internal modification enzymes. This paper reviews the structure, function, interaction with other catalytic domains, and the reduction of recombinant module activity of PCP, aiming to provide a theoretical basis for the construction of recombinant NRPS modules.
    Citation
    ZHU Chenyang, WU Xinyuan, XUE Yongchang.Advances in structure and function of peptidyl carrier protein[J].Microbiology China,2022,49(7):2778~2788
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  • Sulfate-reducing bacteria (SRB), key members of the gut microbiota, produce hydrogen sulfate (H2S) during growth and metabolism. Studies have demonstrated that the overgrowth of SRB is associated with inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), celiac disease (CLD), and colorectal cancer (CRC). However, there is no relevant research review to systematically address the role of SRB in intestinal diseases, pathogenesis, and intestinal microenvironment. Therefore, the characteristics of SRB in intestinal diseases and its pathogenic mechanism deserve to be further summarized and explored. This review collected the literature on SRB-related intestinal diseases published in the past decade and conducted a detailed analysis and summary. The research progress and other aspects were discussed in detail to increase the attention of researchers in this field to SRB. Moreover, we pointed out several areas that need in-depth research for understanding the role of SRB in intestinal diseases. We hope that this review will serve as a valuable reference for the treatment and prevention of SRB-associated diseases in the foreseen future.
    Citation
    HUANG Guoxin, WANG Jingyi, ZHENG Yilin, LI Qingnan, SU Jiyan, HSIAO W. L. Wendy.The association between sulfate-reducing bacteria and gut-related diseases: a review[J].Microbiology China,2022,49(7):2789~2804
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  • Nitrogen cycle within composting is largely dependent on microorganism-driven nitrogen transformation. However, the maximum temperatures of conventional thermophilic composting systems only reach 55–60 ℃. Notably, rising the temperature can shorten the duration of processing and improve the quality of end products. Hyperthermophilic composting as an innovative technology has been developed in recent years, breaking through the limitations of low pile temperatures. The continuous ultra-high temperature regulates the interactions between composting microbiome, composting environment, and nitrogen, and thus significantly reduces nitrogen loss. This paper introduced the basic process of nitrogen transformation and the superiority in nitrogen retention during hyperthermophilic composting. Further, we summarized the recent research progress in dominant microbial groups associated with nitrogen transformation, the factors influencing functional microbes, and the mechanism for controlling nitrogen loss in hyperthermophilic composting. Finally, we analyzed the problems in the current research about hyperthermophilic composting and discussed the possible solutions.
    Citation
    LU Xiaolin, ZHU Weijing, WANG Jingbang, WANG Weiping, ZHU Fengxiang, HONG Chunlai, HONG Leidong, YAO Yanlai, QI Xingjiang, ZHOU Wenlin.Progress in the microbial mechanism for the promotion of nitrogen loss reduction by hyperthermophilic composting[J].Microbiology China,2022,49(7):2805~2818
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  • Plant wilt caused by Fusarium oxysporum is a soil-borne disease common in agricultural production systems and difficult to control, which possess a serious threat to crop yield and quality. Arbuscular mycorrhizal (AM) fungi can form mutualistic symbiosis with most terrestrial plants and play important roles in promoting plant growth and enhancing plant disease resistance. We collected and analyzed the relevant literature published during 2001–2021 and summarized the research progress of biological control of F. oxysporum-caused wilt by AM fungi. We investigated the effects of AM fungal inoculum compositions and application modes on the disease development and the abundance of F. oxysporum. We further summarized the direct and indirect mechanisms of AM fungi in enhancing plant resistance against F. oxysporum from the aspects of soil, root and plant. The mechanisms included affecting soil micro-environment, regulating rhizosphere microbial community structure, competing for niche with pathogens, strengthening root mechanical protection barrier, promoting nutrient absorption and inducing systemic resistance of plants. In addition, we analyzed the progress and deficiency of AM fungi combined with other measures for the control of F. oxysporum-caused wilt. The present review is expected to promote the development of fundamental research and application of AM fungi in biological control of plant diseases.
    Citation
    WANG Haixi, HAO Zhipeng, ZHANG Xin, XIE Wei, CHEN Baodong.Effect, mechanisms and application of arbuscular mycorrhizal fungi for biological control of Fusarium oxysporum-caused wilt: a review[J].Microbiology China,2022,49(7):2819~2837
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  • Chinese herbal medicines may be contaminated with microorganisms (pathogenic bacteria and mycotoxigenic fungi) during cultivation, harvesting, processing, and storage, which causes spoilage and consequently affects the safety of herbal medicines. This study summarized the microbial contamination of common herbal medicines in recent years, and compared the limits of microbial and mycotoxin in pharmacopeias of different countries. Moreover, we reviewed the detection and identification methods of microorganisms in herbal medicines to provide a reference for similar research.
    Citation
    CHEN Ling, CHEN Wei, LIU Zhenjie, ZENG Jiahui, GUO Weipeng, WU Qingping, ZHANG Jumei.Progress on contamination and identification methods of harmful microorganisms in Chinese herbal medicines[J].Microbiology China,2022,49(7):2838~2848
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  • The messenger RNA (mRNA) platform has emerged as a novel nucleic acid technology for the development of vaccines and antibodies in the last decade. mRNA vaccines are superior to conventional vaccines because of safe administration, high potency, short development cycle and low cost of manufacturing. The mRNA-encoded antibodies prevail over other antibody expression platforms because of the high level and long duration of protein expression. Owing to the recent innovations in mRNA modification and delivery, mRNA platform has been developing rapidly and become a promising tool in vaccine development and cancer therapy. It is a miraculous scientific triumph to develop novel mRNA vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which paves the way to a promising future of this field. Here, we overview the ongoing efforts for the optimization of mRNA approaches, with an emphasis on the research progress of mRNA vaccines and mRNA-encoded antibodies for infectious diseases. Furthermore, we put forward the key issues facing the mRNA platform in combating infectious diseases.
    Citation
    TIAN Ying, ZHANG Nana, QIN Chengfeng, LI Xiaofeng.Advancements in mRNA technology-based therapies for infectious diseases[J].Microbiology China,2022,49(7):2849~2861
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  • LI Liang, GUO Nannan, HAO Ruiying, CHANG Lele
    Microbiology China. 2022,49(7):2862-2874
    DOI: 10.13344/j.microbiol.china.211046
    Piriformospora indica, an endophytic fungus, colonizes the roots of various plant species to promote their growth, nutrient absorption, accumulation of secondary metabolites, and tolerance to biotic and abiotic stresses and increase their biomass. Therefore, P. indica has great potential for biocontrol and soil improvement and thus can be applied in agricultural production. In this paper, we summarized the research progress of P. indica in improving the resistance of plants to biotic and abiotic stresses in recent 10 years, hoping to enhance the understanding P. indica-plant interaction and bring into full play its functions.
    Citation
    LI Liang, GUO Nannan, HAO Ruiying, CHANG Lele.Research progress of Piriformospora indica in enhancing stress resistance of plant[J].Microbiology China,2022,49(7):2862~2874
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  • [Background] Fecal microbiota transplantation is a hot research topic in medical field in recent years. It can not only treat digestive system diseases but also have significant efficacy for neuropsychiatric disorders and cardiovascular diseases. [Objective] To learn the latest research status, hot spots, development trends of fecal microbiota transplantation and to provide reference for researchers in related fields. [Methods] CiteSpace was employed to conduct the visualized analysis of annual publications, authors, countries, journals, citations and keywords of the related papers from 2011 to 2021 in the Web of Science Core Collection. [Results] A total of 4 905 papers were included for analysis after screening. The annual publications increased rapidly, and the United States and China were the top contributors. Although the total number of papers published by Chinese researchers ranks the second in the world, the papers had low centrality and citation frequency, which indicated low quality and weak academic influence. Gastroenterology was the journal with the largest number of articles published, and Frontiers in Microbiology was the journal with the largest number of articles published by Chinese researchers. Fecal microbiota transplantation presented the trend of interdisciplinary development. The current studies about fecal microbiota transplantation mainly focused on intestinal diseases (e.g., inflammatory bowel disease and Clostridium difficile infection) and extragastrointestinal diseases (e.g., depression and coronary atherosclerosis). The application of fecal microbiota transplantation in juveniles, influence of fecal microbiota transplantation on insulin sensitivity, application of sequencing technology in intestinal flora research, and more accurate therapies with fecal microbiota transplantation may become the hot spots in the future. [Conclusion] The research on fecal microbiota transplantation is advancing rapidly, with the focus gradually expanding from intestinal diseases to extragastrointestinal diseases. It is an inevitable trend to accurately apply fecal microbiota transplantation to the treatment of other diseases in the future.
    Citation
    YIN Shuoxin, ZHANG Tao, LU Xin, WANG Shuping, HUANG Xuping, YIN Mengying, YANG Yuwei, MAO Zhihua, CHEN Yuanneng.Bibliometric and visualized analysis of research on fecal microbiota transplantation[J].Microbiology China,2022,49(7):2875~2887
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Microbiology China Monthly

ISSN: 0253-2654
CN: 11-1996/Q
Start Publication in 1974 Released on the 20th per month Responsible institution: Chinese Academy of Sciences Sponsored by: Institute of Microbiology, CAS Chinese Society for Microbiology Editor-in-Chief: HE Rong-Qiao Editorial office: Tel: +86-10-64807511 E-mail: tongbao@im.ac.cn