Abstract:[Background] The glycoside hydrolase 3 gene (GH3) family mainly encodes extracellular β-glucosidases, which are the key enzymes in cellulose degradation. [Objective] To identify the GH3 genes in Trichoderma asperellum (TaGH3 genes) and investigate their transcriptional patterns. [Methods] The bioinformatics tools were employed to identify the TaGH3 genes and analyze the gene structure, phylogenetic relationship, as well as the physicochemical properties, subcellular localization, and tertiary structure of the deduced proteins. quantitative real-time polymerase chain reaction (RT-qPCR) was employed to investigate the transcriptional patterns of TaGH3 genes in the presence of cellulose. [Results] A total of 16 TaGH3 genes were identified, which contained 1-8 exons. The deduced TaGH3 proteins had the length of 533-934 amino acid (aa) and the theoretical molecular weights of 57.82-101.91 kDa. The majority of TaGH3 proteins were predicted to be extracellular. Phylogenetic analysis demonstrated that all the TaGH3 proteins could be classified into 4 clades, which had higher homology with Trichoderma reesei. Cellulose affected the transcription of all the 16 TaGH3 genes, while the transcription level varied among genes. Specifically, cellulose induced the constitutive expression of 1 gene, down-regulated the expression of 2 genes, and up-regulated the expression of 13 genes. Moreover, cellulose enhanced the extracellular β-glucosidase activity, which was consistent to the transcriptional pattern of most TaGH3 genes. [Conclusion] The genome of T. asperellum carried 16 GH3 genes, most of which encoded hydrophilic proteins with the potential for commercial exploitation. The transcription of the majority of TaGH3 genes was up-regulated by cellulose, which was consistent with the enhanced β-glucosidase activity, indicating that these genes played a key role in cellulose degradation. This study systematically investigated the GH3 genes in T. asperellum, providing new insights for the resource utilization of lignocellulosic materials and the construction of strains with high cellulase production.

Abstract:[Background] Androsace tapete is the representative cushion plant species on the Qinghai-Tibetan Plateau, and the phyllospheric and intrafoliar microorganisms are important for this species to adapt to the extreme environment and are also a unique resource. [Objective] To investigate the diversity of phyllospheric and endophytic culturable microorganisms in A. tapete and the differences in the microorganisms between living and dead individuals. [Methods] Phyllospheric and intrafoliar bacteria, yeasts, and filamentous fungi were isolated from A. tapete in three different regions by pure culture, purified, and identified based on 16S rRNA and ITS sequence analysis. [Results] A total of 350 phyllospheric strains (49 species in 22 genera) were identified, with the dominant species of Penicillium sajarovii, and 274 endophytic culturable strains (45 species in 19 genera) were screened out, with the dominant species of Bacillus mycoides. The two shared the dominant Penicillium. The difference in alpha diversity between phyllospheric and intrafoliar microorganisms of A. tapete and between the living and dead individuals was mostly not significant. The communities shared some members, but there was significant spatial heterogeneity in species composition. [Conclusion] There are abundant culturable phyllospheric and intrafoliar microbial resources in A. tapete, and the species composition was remarkably different between living and dead individuals and between different parts of the leaf, and the selection preferences of microorganisms for different environments result in different community patterns. However, the microbial communities from different sources shared a high proportion of strains, and the heterotrophic mode and ecological niche of these shared strains are not fixed. The heterotrophic pattern and the niches of the shared strains are not fixed. They can survive by both symbiosis and saprophytism, and the living space has no clear boundary. The purified strains can be used for the development and utilization of functional strains.

Abstract:[Background] Gansu Baiyin Copper Mine is an important mining area in western China. It is of great significance to explore the diversity and slope distribution of bacteria and archaea in the area. [Objective] To analyze the community diversity of bacteria and archaea in soil samples at different slopes of Baiyin Copper Mine pit and further the relationship between the diversity and physico-chemical properties of soils. [Methods] Illumina HiSeq was employed to elucidate the diversity of bacteria and archaea in soil samples at different slopes of the pit. [Results] Alpha and beta diversity indices of ACE index, Chao1 index, Shannon index, and Simpson index showed significantly different among samples. The top ten classes of bacteria in the pit included Gammaproteobacteria, Oxyphotobacteria, Alphaproteobacteria, Bacteroidia, Actinobacteria, Clostridia, Nitrospira, Bacilli, Mollicutes, and Acidimicrobiia, among which Gammaproteobacteria was most abundant. At the class level, the archaea of the pit were composed of Thermoplasmata, Nitrososphaeria, Methanomicrobia, Halobacteria, Methanobacteria, Group_1.1c, and Bathyarchaeia, and the abundance of Thermoplasmata was the highest. Redundancy analysis (RDA) suggested that pH and available phosphorus were the major factors affecting soil diversity. [Conclusion] Baiyin Copper Mine is a strong acid mine. The content of copper ions was the highest in soil of Baiyin Copper Mine pit. The pit registered the pH trend of first decreasing and then increasing (2.18-4.75), trends of copper ions (20-150 mg/kg) and total iron (2.5-50 mg/kg) opposite to pH, low contents of ammonium nitrogen (3.61-44.90 mg/kg), available phosphorus (0-56.79 mg/kg), and available potassium (9.07-35.65 mg/kg) with fluctuation, and no obvious variation of sulfide (1 mg/kg) from the surface to the bottom. At the genus level, Acidithiobacillus was most abundant among the bacteria, while the counterparts for archaea were uncultured_bacterium_ f_Ferroplasmaceae and uncultured_bacterium_ f_ Nitrososphaeraceae. pH and available phosphorus were the main factors causing the variation of bacterial and archaeal diversity, respectively. This study is expected to deepen the understanding of changes in soil microbial communities in Baiyin Copper Mine.

Abstract:[Background] Phyllosphere is home to various and abundant microorganisms. Thanks to the specific functions, phyllosphere microorganisms survive in the presence of stresses and they influence the physiological and ecological characteristics of host plants. In addition, they are affected by environmental heterogeneity. [Objective] The microbial community in plant phyllosphere is dynamic, and clarifying the effect of seasonal alternation on structure of the microbial community is of great significance for in-depth understanding of plant-microbe-environment interactions. [Methods] Reaumuria trigyna is a recretohalophyte in desert steppe in Ordos. We determined the physico-chemical properties of leaf surface and carried out high-throughput sequencing of phyllosphere bacteria and fungi in spring and autumn. [Results] The water content, pH, and electric conductivity of soil under the canopy of R. trigyna and the electric conductivity, Na⁺ content, and K⁺ content on leaf surface were significantly different between spring and autumn. The operational taxonomic units (OTUs), Shannon, Chao1 and ACE indices of phyllosphere bacteria were in positive correlation with salt content of soil and leaf surface. The relative abundance of Cyanobacteria and Bacteroidota was higher in spring, while the relative abundance of Proteobacteria, Actinobacteriota, and Ascomycota in autumn was higher than that in spring. Bradyrhizobium, Novosphingobium, and Edaphobaculum were in positive correlation with leaf surface salinity, while Modestobacter, Adhaeribacter, and Bacillus showed negative correlation. [Conclusion] The asynchronous change of water and salt in soil under the canopy of R. trigyna caused by seasonal alternation increased the accumulation of salt on the leaf surface of R. trigyna, further affecting the phyllosphere microbial diversity and community composition. This study reveals the response of phyllosphere bacteria and fungi of R. trigyna to seasonal alternation, which is expected to provide a theoretical reference for further elucidating the assembly strategies of phyllosphere microbial communities under stress.

Abstract:[Background] The investigation of Cordyceps species diversity in different areas is one of the hotspots of Cordyceps -related research, and the study on Cordyceps -related fungi flora is an important content of fungus resources research. [Objective] To investigate the diversity of natural entomogenous fungi and endophytic fungi in Xishan Forest Park of Kunming in Yunnan, and increase the understanding of Cordyceps -related fungi flora. [Methods] The resources of Cordyceps species were investigated, and the tissue separation method was used to isolate entomogenous fungi and endophytic fungi from the samples of Cordyceps species. Morphological observation and multi-gene sequencing of ITS combined with nrSSU, nrLSU, translation elongation factor-1α (tef-1α), rpb1, and rpb2 were used to identify the entomogenic fungi of Cordyceps species and analyze their diversity. [Results] Forty-five natural samples of Cordyceps species were collected from Xishan Forest Park of Kunming, which belonged to 12 species, 5 genera, and 2 families, including 2 Samsoniella sp. with 1 potential new species, 4 Cordyceps sp., and 4 Beauveria sp. in Cordycipitaceae, and 1 Purpureocillium sp., and 1 Tolypocladium sp. (potential new species) in Ophiocordycipitaceae. The dominant genus was Cordyceps, and the dominant species was Cordyceps cateniannulata (ecological dominance=0.241 380). Meanwhile, 67 strains of endophytic fungi in 14 genera of 13 families were isolated from the samples, among which Trichoderma (28%) and Fusarium (19%) were the dominant genera. [Conclusion] This study supplements the resource database of Cordyceps biodiversity in Xishan Forest Park of Kunming, which is conducive to the protection and utilization of Cordyceps biodiversity resources in Yunnan. At the same time, this systematic study also deepens the understanding of Cordyceps -related fungi flora.

Abstract:[Background] Decabromodiphenyl ether (BDE-209) is one of the most widely used brominated flame retardants, which has caused serious harm to ecological environment and organisms due to its persistent toxicity. [Objective] To obtain an aerobic bacterium that could effectively degrade BDE-209 and to study its degradation characteristics and mechanisms. [Methods] The aerobic degrading bacterium was isolated from activated sludge by enrichment, isolation and purification using BDE-209 as the sole carbon source. The concentration of BDE-209 and the intermediate products were analyzed by high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS), respectively. [Results] A BDE-209 aerobic degrading strain F was screened and preliminarily identified as Pseudomonas nitroreducens. Under the optimal degradation conditions of 30℃, pH 7.0, 10% inoculum volume and 250 mg/L glucose, the degradation efficiency reached 76.2% for 10 mg/L (initial concentration) BDE-209. Obvious effects of low Cd²⁺concentrations (≤5 mg/L) on BDE-209 biodegradation and the growth of P. nitroreduc ens were not observed, while the effects of high Cd²⁺concentrations (≥15 mg/L) were significant. In addition, BDE-209 affected cell surface hydrophobicity to some degree. The biodegradation mechanisms of BDE-209 mainly included debromination, hydroxylation, cleavage of diphenyl ether bonds and ring opening. [Conclusion] Strain F showed good biodegradation ability for BDE-209. The research results facilitated the aerobic microbial degradation of BDE-209 and the bioremediation of BDE-209 polluted environment.

Abstract:[Background] Some members of the genus Pseudoalteromonas can secrete a variety of chitinases, which play important roles in degrading chitin to provide nutrition, immunity, and disease prevention for aquatic animals. [Objective] To clone a chitinase gene of Pseudoalteromonas piscicida C923 and realize its heterologous expression in Escherichia coli, thereby exploring the enzymatic properties of the recombinant chitinase. [Methods] A potential gene PpchiC, identified and annotated as chitinase based on the analysis of the genome sequence of strain C923, was cloned by designing primers and subsequently subjected to bioinformatic analysis. Then an expression vector was constructed to conduct heterologous expression, and the expression was optimized from the temperature, time, and concentration of the inducer. The optimum temperature, pH of the expressed protein, and other enzymatic properties were studied. Finally, the degradation of chitin by the supernatant and precipitation of recombinant E. coli cells and the purified enzyme protein were compared. [Results] PpchiC was 1 350 bp in length and encoded 450 amino acids. The theoretical molecular mass of protein PpchiC was 48.76 kDa, and its isoelectric point and instability coefficient were 4.78 and 29.08, respectively. Structural analysis revealed that PpchiC contained a type III chitin-binding domain, a catalytic domain of glycosyl hydrolase 18 (GH18), and conserved motifs DxxDxDxE, YxR, and [E/D]xx[V/I]. The optimized expression conditions were 16 ℃ of temperature, induction time of 12 h, and IPTG concentration of 0.25 mmol/L. PpchiC showed maximum enzymatic activity at 50 ℃ and pH 8.0. When colloidal chitin was used as the substrate, the kinetic parameters K _m and V _max were 2.58 mg/mL and 5.04 mg/(mL·min), respectively. The results of degradation showed that the supernatant and precipitation of recombinant E. coli cells and the purified enzyme protein from the supernatant all exhibited excellent chitin degradation effects. [Conclusion] The gene PpchiC from P. piscicida C923 encodes a GH18 family chitinase, which can be highly expressed in E. coli with obvious chitin degradation effects. This study provides references for the application of Ppchic and C923 strain.

Abstract:[Background] Bacillus safensis ST7 has strong ability of oxidizing manganese, the molecular mechanism of which, however, remains unclear. [Objective] The aim is to study the effect of the flagellar motor switch protein FliY on the manganese-oxidizing ability of B. safensis. [Methods] Based on the principle of homologous recombination, a fliY -deleted mutant (△fliY) was constructed using B. safensis ST7 as the starting strain. The changes in mobility, biofilm formation, and manganese-oxidizing capacity of △fliY were evaluated based on colony migration, biofilm production, and Mn(II)-oxidizing rate. [Results] After cloning and sequencing, it was confirmed that fliY was inactivated with the last part replaced by kanamycin resistance gene in △fliY. The growth of △fliY in LB medium was similar to that of the starting strain. However, △fliY showed retarded logarithmic growth, small colonies, and reduced biofilm production in the PYCM medium containing Mn(II). The motility and Mn(II)-oxidizing rate of △fliY decreased by about 65% and 20%, respectively. [Conclusion] The gene fliY not only affects the growth and motility but also participates in biological processes such as chemotaxis and manganese oxidation of B. safensis.

Abstract:[Background] A few studies of the effect of rotation system on the community structure and diversity of soil nitrogen-fixing microorganisms on plateau are available. [Objective] To clarify the effect of rotation system on farmland soil fertility and diversity and community structure of soil microorganisms with nifH gene on Panxi Plateau and screen a reasonable rotation system. [Methods] Soil samples were collected from five crop rotation systems: 15-year Vicia villosa Roth-flue-cured tobacco rotation (G1), 20-year V. villosa -flue-cured tobacco rotation (G2), tartary buckwheat (Fagopyrum tataricum)-flue-cured tobacco rotation (KQ), barley (Hordeum vulgare L.)-flue-cured tobacco rotation (DM), and abandoned soil (CK) in Mianning county, Liangshan prefecture. Through chemical analysis and high-throughput sequencing of nifH, soil physico-chemical properties and community structure and diversity of soil nitrogen-fixing microorganisms were analyzed. [Results] The total nitrogen content, ammonium nitrogen content, nitrate nitrogen content, organic carbon content, and water content were the highest in the CK (P <0.05) and the pH value was below 7 in all rotation systems. Under KQ, content of soil available phosphorus and available potassium was 43.0% and 2.60% higher, respectively. Soil physico-chemical parameters were lower under DM rotation than in CK. Soil nitrogenase activity was highest in CK and lowest under G2. The diversity of nitrogen-fixing microorganisms was the highest under G1 and lowest under G2. At the phylum level, the relative abundance of Proteobacteria was the highest, accounting for 63.0%-92.4% of the total abundance. At genus level, Azohydromonas dominated the soils under crop rotation and Bradyrhizobium was most abundant in CK. Redundancy analysis revealed that soil pH and water content were the key factors driving the composition of soil nitrogen-fixing microorganisms. [Conclusion] Rotation systems obviously changed the soil fertility and the composition of nifH gene community, particularly the G1 which improved soil nutrient content and diversity of soil nitrogen-fixing microorganisms.

Abstract:[Background] With strong bacteriostatic activity, Gram-positive Bacillus subtilis N2-10 produces diverse hydrolases such as cellulase, thus showing huge potential in feed fermentation. [Objective] The whole genome of N2-10 was sequenced and the genes related to the synthesis of the secondary metabolites were analyzed. Based on comparative genomics, the difference between N2-10 and the model strain was dissected. The findings are expected to lay a theoretical basis for clarifying the bacteriostatic and probiotic mechanisms of this strain. [Methods] The genome of N2-10 was sequenced by Illumina NovaSeq and PacBioSequel, followed by genome assembly, gene prediction, and functional annotation. The differences between N2-10 and a model strain were analyzed by comparative genomics. [Results] The genome of N2-10 is 4 036 899 bp, with GC content of 43.88%. It has 4 163 coding genes with the total length of 3 594 369 bp, and the total length of the coding region accounts for 89.1% of the total genome length. In detail, it has 85 tRNA genes, 10 5S rRNA genes, 10 16S rRNA genes, 10 23S rRNA genes, 2 CRISPR-Cas sequences, and 6 gene islands. Moreover, this strain was also found to have a prophage. In addition, 3 048, 3 177, 3 894, and 145 genes were annotated in GO (gene ontolog), COG (clusters of orthologous groups of proteins), KEGG (Kyoto encyclopedia of genes and genomes), and CAZy (carbohydrate-active enzymes) databases, respectively. At the same time, 10 gene clusters for the synthesis of secondary metabolites were predicted, including biosynthesis gene clusters rhizocticin A, bacillaene, fengycin, bacillibactin, subtilosin A, and bacilysin and 3 unknown gene clusters. Comparative genomics analysis showed that N2-10 had high homology with B. subtilis 168. [Conclusion] Through genome sequencing, we unveiled the genetic information of N2-10, providing a reference for further understanding the secondary metabolites of this stain and further development and utilization of it.

Abstract:[Background] Melatonin is a small-molecule biogenic amine ubiquitous in animals and plants, which promotes the growth and environmental adaptation of organisms. Trichoderma act as important biocontrol strains and engineering strains of industrial products, which can produce diverse secondary metabolites. [Objective] Since the melatonin synthesis in Trichoderma strains remains unclear, we constructed a Trichoderma viride strain capable of synthesizing melatonin and characterized its physiological properties. [Methods] In this study, the genes hAANAT encoding human alkylamine N-acetyltransferase and hASMT encoding human acetylserotonin O-methyltransferase were heterologously expressed in T. viride Tv-1511. The yield of melatonin synthesized by the engineered strain was determined by high performance liquid chromatography (HPLC). Furthermore, the growth, stress resistance, and growth-promoting and biocontrol abilities of the engineered strain were determined by biochemical methods. [Results] A melatonin-producing strain of T. viride was obtained, which had better growth and sporulation characteristics, stronger stress tolerance, stronger antagonism to pathogens, and higher plant growth-promoting ability than the start strain. [Conclusion] Melatonin plays a positive role in the growth and stress resistance of T. viride. It is of great significance to construct the Trichoderma strains with melatonin-synthesizing ability by genetic modification.

Abstract:[Background] High temperature decreases microbial activity, thus limiting the lignin degradation in garden waste composting. [Objective] To acclimatize a lignin-degrading strain—Bacillus sp. NO.2, improve its activity at high temperature, and observe its growth status and enzymatic properties. [Methods] The temperature gradient method was used to domesticate the strain, and the growth curve, enzyme activity, and lignin degradation rate were compared before and after the domestication. Moreover, the temperature range and pH range for the enzymatic reaction of lignin-degrading enzymes generated by the strain were determined. [Results] The maximum biomass was insignificantly different between the original strain and the domesticated strain at 60 ℃. However, the activity of laccase (Lac), manganese peroxidase (MnP), and lignin peroxidase (LiP) was 30.75%, 35.98%, and 29.62% higher, and the lignin degradation rate was 60.52% higher than those of the original strain, respectively. The Lac, MnP and LiP produced by the domesticated strain showed high activity at 20-60 ℃ and pH 3.0-9.0, and were stable, with the stability following the order of Lac>LiP>MnP. [Conclusion] The temperature gradient method can improve the activity of Bacillus at high temperature and widen the temperature and pH ranges for enzymatic reaction of lignin-degrading enzymes. Furthermore, it has potential for developing a special microbial agent for degrading garden waste.

Abstract:[Background] Endophytic bacteria of medicinal plants can produce the same or similar compounds as host plants and new secondary metabolites, which can promote the growth of the host, resist diseases and insect pests, and degrade toxic and harmful compounds. [Objective] To further improve the biocontrol efficiency of apple Valsa canker, and enrich the resource pool of antagonistic strains of endophytic bacteria from medicinal plants in Xinjiang. [Methods] The pathogens of apple Valsa canker were isolated and identified from the branches with disease spots of the apple plants in the wild apple forests of Xinyuan County (Yili area) and Emin County (Tacheng area) in Xinjiang. The endophytic bacteria of medicinal plants, which had the ability against the pathogen of apple canker, were screened by plate confrontation method. [Results] A total of 234 strains of pathogenic fungi were isolated from Xinyuan county and Emin county, including 25 strains of Valsa malicola and 2 strains of V. mali. In addition, 92 strains of endophytic bacteria with antifungal effect were screened out, among which 70 strains were from Glycyrrhiza uralensis. [Conclusion] The medicinal plant G. uralensis is rich in microbial resources against apple Valsa canker. This study is of great significance for the biocontrol of apple Valsa canker in Xinjiang wild apple forests and the development and utilization of endophytic bacteria in medicinal plants.

Abstract:[Background] Nosema ceranae exclusively infects midgut epithelial cells of adult bees, and the resulting microsporidiosis causes severe losses to the beekeeping industry. [Objective] This study aimed to determine the expression profiles of nce-miR-23928 and its target genes during the N. ceranae infection of Apis mellifera ligustica workers, and to provide basis for further investigation on the function and regulatory mechanism of nce-miR-23928 during the infection. [Methods] Target genes of nce-mir-23928 were predicted by RNAhybrid, miRanda and TargetScan. Blast was used to perform annotation of the aforementioned target genes in gene ontology (GO), kyoto encyclopedia of genes and genomes (KEGG), Nr and Swissprot. Real time quantitative PCR (RT-qPCR) was employed to detect relative expression of nce-miR-23928 and its target genes. [Results] As compared with the condition at 1 day post infection (1 dpi), the expression of nce-miR-23928 remained unchanged at 2 dpi, but was all down-regulated at 4 6, 8 dpi (P <0.05), presenting an overall reduced expression trend. Additionally, 15 target genes of nce-miR-23928 were predicted, among which 9, 4, 15 and 9 were annotated in GO (3 items), KEGG (7 pathways), Nr and Swiss-prot, respectively. As compared with the condition at 1 dpi, the expression of target gene ABCT was significantly down-regulated at 2, 4, 6, 8 dpi, while the expression of target gene STPK was significantly up-regulated at 4, 6, 8 dpi, displaying an overall elevation trend. [Conclusion] These results illuminated the dynamic expression rules of nce-miR-23928 and its target genes ABCT and SPTK in the N. ceranae infection of A. mellifera ligustica workers, and unraveled that nce-miR-23928 putatively modulated the infection process through positively regulating the expression of ABCT and negatively regulating the expression of STPK.

Abstract:[Background] Low temperature is the main obstacle factor affecting the stable and high yield of tomato in solar-energy greenhouse in northern China. We found that Streptomyces sp. TOR3209 improved the cold tolerance of tomato plants. [Objective] To explore whether the impact of strain TOR3209 on improving resistance to cold stress of tomato plants is related to their endophytic bacterial community structure and property. [Methods] Tomato roots were inoculated with strain TOR3209 in substrate culture, and cultured under low temperature (5 ℃) and mild temperature (25 ℃) separately. Traditional culture-dependent methods were used to isolate and identify bacteria, and the effects of strain TOR3209 on the composition and structure of the root and stem bacterial community were analyzed by 16S rRNA gene amplicon sequencing technology. [Results] In this study, 69 species of endophytic bacteria belonging to 28 genera, 20 families and 4 phyla were isolated. Compared to the conditions in the control group, the Shannon index and Margalef index of the endophytic bacterial community of tomato roots and stems inoculated with strain TOR3209 were increased under low and mild temperatures. At phylum level, the relative abundance of Firmicutes was increased, while that of Proteobacteria and Bacteriodetes was decreased; at genus level, the relative abundance of Bacillus was boosted, while that of Pseudomonas and Flavobacterium was reduced; at species level, after treatment with strain TOR3209, the relative abundance of Bacillus amyloliquefaciens, Bacillus velezensis, and Bacillus aryabhattai was elevated significantly. Under both temperatures of 5 ℃ and 25 ℃, Bacillus wiedmannii, Brevibacterium frigoritolerans and Acinetobacter johnsonii were isolated from the tomato plants inoculated with strain TOR3209, and the experiment demonstrated that the three bacteria are plant-beneficial and cold-adapted, and can markedly increase the heights, leaf areas and biomasses of tomato plants; especially B. frigoritolerans treatment had the most obvious effect on the biomass. Compared with the conditions in the control group, fresh weight of root and shoot rose by 55.13% and 51.03%, respectively, and dry weight rose by 48.37% and 50.95%, respectively. The pathogenic bacteria such as Pantoea agglomerans, Pseudomonas mediterranea and Pseudomonas corrugate were not isolated. [Conclusion] Inoculation with strain TOR3209 impacted the composition and structure of the bacterial community in tomato roots and stems. The relative abundance of indigenous cold-adapted and beneficial microorganisms was increased, and the pathogenic bacteria was decreased, which improved plant cold tolerance.

Abstract:[Background] Lespedeza daurica is an important native grass for the restoration and improvement of degraded salt-alkali grassland in the Loess Plateau. Plant-endophyte symbiosis play an important role in maintaining and improving plant stress resistance. [Objective] To screen out the dominant endophyte with salt-alkali resistance and evaluate their basic characteristics, salt-alkali tolerance, and growth promoting properties. [Methods] A saline-alkali resistant endophyte strain DP20 was screened out from the root system of L. daurica artificially cultured in Shanxi Province. DP20 was identified by morphological observation, physiological and biochemical measurements, and 16S rRNA gene sequencing analysis. The liquid mediums with different salt-alkali concentration (salt concentration was 0, 1%, 3%, 5%, and 7%, and the pH was 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0) were used to measure their saline-alkali tolerance. The functional mediums were used to measure their growth-promoting function. Meanwhile, the catalyst was quantitatively determined. The petri dish filter paper method was used to verify the effects of this strain on the seed germination and growth of L. daurica. Plate test was used to explore the effects of this strain on the growth of Arabidopsis thaliana. [Results] The strain DP20 was Enterobacter ludwigii. The optimum salt concentration for its growth was 0-5%, and the highest tolerance concentration was 7%. It was found that strain DP20 had the ability to produce auxin (IAA), up to 30.81 mg/L, and the decomposition capacity of organophosphorus, reaching 0.53 mg/L. Furthermore, the strain had the ability to produce siderophores and secrete organic acids, and the concentration of siderophores reached 67.67%. Strain DP20 grew on the NFM solid medium. Strain DP20 significantly promoted the seed germination rate, plant fresh weight, and root development of L. daurica and A. thaliana. [Conclusion] The saline-alkali tolerant strain DP20 has significant growth promoting ability, which provides efficient and saline-alkali resistant endogenous resources for the later application of L. daurica in the improvement of saline-alkali soil in northern Shanxi, and is helpful to develop the potential of E. ludwigii as a microbial agent.

Abstract:[Background] Trichoderma is a kind of fungus widely distributed in nature, which produces a variety of enzymes and secondary metabolites, showing many benefits including promoting plant growth, improving soil fertility, and antagonizing various soil-borne pathogens. [Objective] To optimize the solid fermentation conditions of three plant growth-promoting fungi (Trichodermalongibrachiatum MD30, T. citrinoviride JS84, and T. guizhouense NJAU4742), the effects of different fermentation conditions on the spore production of Trichoderma spp. were explored to provide references for Trichoderma spp. production. [Methods] In this study, the spore production of three Trichoderma spp. under different fermentation conditions was measured and optimized by the single factor experiment and the response surface methodology. The effects of the addition of nitrogen source, initial pH, material thickness, inoculation amount, and temperature on solid fermentation were analyzed. [Results] The single factor experiment showed that the optimal fermentation temperature of T. longibrachiatum MD30, T. citrinoviride JS84, and T. guizhouense NJAU4742 was 28 ℃, and the optimal amount of Trichoderma liquid inoculum was 10% with 3.0 cm material fermentation thickness. However, the optimal initial pH of the material and the addition amount of amino acid hydrolyzate were different among these three strains. In detail, the optimal initial pH value of T. longbranchus MD30 and T. guizhouense NJAU4742 for solid fermentation was 5.0, while that of T. citrinoviride JS84 was 3.0. The optimum amino acid hydrolyzate addition amount for solid fermentation of T. longbranchus MD30 and T. guizhouense NJAU4742 was 10%, while that of T. citrinoviride JS84 was 5%. Based on the experimental analysis, the initial pH, material thickness, and temperature were selected as three top important factors in the determination of the spore production. Therefore, the optimal fermentation conditions for solid fermentation of these three strains were obtained based on the response surface analysis. The optimal conditions for solid fermentation of T. longbranchus MD30 were the initial pH value of 5.0, the material thickness of 3.0 cm, and thetemperature of 27.4 ℃. Under this optimized condition, the number of spores per gram dry weight of the solid fermentation product was as high as 6.3×10⁹ CFU. The optimal conditions for solid fermentation of T. citrinoviride JS84 were the initial pH value of 3.0, the material thickness of 3.0 cm, and the temperature of 28.8 ℃. Under this optimized condition, the number of spores per gram dry weight of the solid fermentation product was as high as 6.9×10⁹ CFU. The optimal conditions for solid fermentation of T. guizhouense NJAU4742 were the initial pH value of 5.0, the material thickness of 3.3 cm, and the fermentation temperature of 26.7 ℃. Under this optimized condition, the number of spores per gram dry weight of the solid fermentation product was as high as 5.1×10⁹ CFU. [Conclusion] Response surface optimization experiment improves the experimental efficiency, increases the spore production of Trichoderma spp., reduces the production cost, and is conducive to field application and commercial production of bacterial agents.

Abstract:[Background] While improving soil fertility and nutrients, straw returning faces the challenge of slow straw degradation and threat of pathogenic fungi. [Objective] To screen efficient straw-degrading strains against the fungi from samples in areas with long-term straw returning. [Methods] The isolation by dilution in liquid, aniline blue staining, and Congo red staining were used to screen the efficient straw-degrading strains and the strains were identified based on 16S rRNA gene sequencing and phylogeny analysis. The inhibition of the screened straw-degrading strains on Setostphaeria turcica, Alternaria kikuchiana, Alternaria solani, and Alternaria alternate aCCC38230 and aCCC38231 was examined by the confrontation assay. Moreover, the ability of the metabolites of the antagonistic strains against S. turcica was determined, and the influence of the strains on conidial germination and hypha growth of S. turcica was detected with crude extract of antagonistic strains. [Results] Three efficient cellulose/lignin-degrading strains were screened out and named as JY122, ZY133, and JY215, which all belong to Bacillus. Phylogeny analysis revealed 99.4% similarity between JY122 and Bacillus cereus, 100% similarity between ZY133 and Bacillus subtilis, and 99.1% similarity between JY215 and Bacillus velezensis. The three had strong inhibitory effect on pathogenic fungi of different species with the inhibition rate in the range of 43.74%-67.54%. In addition, the metabolites of them showed antifungal activity and strong thermal stability, as the antifungal activity was still high after treatment at 95 ℃. [Conclusion] JY122, ZY133, and JY215 show high efficiency in degrading cellulose/lignin and inhibit the growth of a variety of plant pathogenic fungi. The metabolites of the three have strong antifungal ability and high thermal stability. This study provides strain resources for returning maize straw and a method for tackling the challenges in straw returning.

Abstract:[Background] Aroma-producing yeast can endow wine with unique aroma. Therefore, the isolation and screening of excellent aroma-producing yeast strains is of great significance for brewing the wine with regional characteristic flavor. [Objective] Aroma-producing yeast strains were screened from the wild grape growing on Zhongtiao Mountain, identified, and characterized for the physiological and biochemical properties. Further, we applied the strain with the strongest aroma to wine fermentation and studied its effect on aromatic components of the wine. [Methods] The aroma-producing yeast strains were isolated with plate-streaking method and then identified based on the molecular evidence. The strain with the strongest aroma was used for mixed fermentation with Saccharomyces cerevisiae F15. The aroma components of the wine were analyzed by gas chromatograph-mass spectrometer (GC-MS). The content of aroma components was determined with semi-quantitative method. [Results] A total of 13 strains was isolated. The D1/D2 region sequence of the 26S rRNA gene indicated that these strains belonged to five genera of Issatchenkia, Torulaspora, Pichia, Saccharomyces, and Rhodotorula. The strain Issatchenkia orientalis XS-6 with the strongest aroma was selected for further study. It could tolerate the highest ethanol concentration of 8% and NaCl concentration of 6% and showcased the most vigorous growth at 38 ℃. A total of 31 aroma components were detected in wine fermented with I. orientalis XS-6 and S. cerevisiae F15. The total content of aroma substances in the wine produced by mixed fermentation increased by 19.8% compared with that by single strain fermentation. The total content of aroma compounds increased by 19.8%, and the content of 11 aroma components, especially phenylethanol with rose aroma, increased significantly. The content of alcohols and esters increased by 19.6%, and seven new esters such as ethyl vanillate and dibutyl phthalate were detected. [Conclusion] The aroma-producing yeast stain XS-6 has good tolerance to ethanol, NaCl, and temperature. Mixed fermentation of XS-6 with S. cerevisiae F15 significantly affects the aromatic components of Syrah wine, which suggests that XS-6 may have a potential application value in improving wine flavor.

Abstract:[Background] Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus are commonly used as yogurt starters, which are widely used in dairy production. [Objective] Based on the previous study, the compound starter with good flavor was screened out (1:100). L. delbrueckii subsp. bulgaricus IMAU20312 and S. thermophilus IMAU80809, and the dynamic variation of flavor compounds in cow milk during the fermentation and storage was analyzed. The result is expected to lay a basis for the development and application of related starters. [Methods] We employed solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) and electronic nose to detect and analyze the dynamic changes of the volatile flavor compounds in milk during fermentation and storage. [Results] A total of 23 acids, 13 aldehydes, 15 alcohols, 18 ketones, 11 esters, and 22 alkanes were produced in the fermentation and storage of milk. According to odor activity values (OAV), there were two key flavor compounds (OAV≥1) in the fermented milk. Moreover, other compounds such as 1-octene-3-ol, 3-hydroxybutyraldehyde, 3-methyl-n-butyraldehyde, 2,3-butanedione, 2-nonanone, and ethyl ester modified the overall flavor of the fermented milk (0.1≤OAV<1). According to the cluster heatmap, the flavor substances produced during fermentation and storage were significantly different. Similar results were also found by electronic nose detection. [Conclusion] The flavor compounds produced by the compound starter in milk during fermentation and storage endow fermented milk a good flavor.

Abstract:[Background] There has been an explosion of research on the germplasm resources, introduction, expansion and breeding, and chemical components of tulip, but no report on the endophytes of wild tulip in Ili is available. [Objective] To screen endophytic fungi from wild tulip in Ili and study their antibacterial and antioxidant activities. [Methods] The endophytic fungi of Ili wild tulip were isolated and purified by tissue culture and streak plate method. The endophytes were preserved with the slant cryopreservation method. The isolated endophytic fungi were identified based on morphological observation and molecular biology methods. The secondary metabolites were obtained by liquid fermentation, and the antibacterial activity of the fermentation product extracted with ethyl acetate was analyzed with the filter paper method. The total Fe³⁺-reducing power, 2,2'-diphenyl-1-picrylhydrazyl (DPPH)-scavenging ability, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonicacid) (ABTS)-scavenging ability, and hydroxyl radical-scavenging ability of the aqueous fraction and ethyl acetate fraction were determined to compare the antioxidant activity. [Results] An endophytic strain was isolated and identified as Aspergillus fumigatus YGL-1. YGL-1 showed significant inhibitory activity against Staphylococcus aureus, Escherichia coli, Bacillus subtilis, and Candida albicans. Both the aqueous fraction and the ethyl acetate fraction can reduce Fe³⁺, and scavenge DPPH, ABTS, and hydroxyl radicals, especially DPPH and ABTS. The antioxidant activity was enhanced with increase in concentration. When the concentration of the crude extract was 2.0 mg/mL, the ethyl acetate fraction can scavenge 91.45% of DPPH and 99.84% of ABTS. In summary, the fermentation products of YGL-1 had strong antioxidant activity, particularly the organic solvent fraction, followed by the aqueous fraction. [Conclusion] A. fumigatus was isolated from wild tulip in Ili. This study is expected to serve as a reference for the comprehensive utilization of endophytic fungi in plant resources and the development of antibacterial and antioxidant drugs.

Abstract:[Background] Gut flora influences the nutrient digestion and absorption, immunity, and behavior of the host and is impacted by the diet, living environment, and other factors of the host. [Objective] Food-provisioning can improve the nutrition in wild animals, but a few reports on the impact on gut flora are available. [Methods] Through high-throughput sequencing of the fresh feces of wild and food-provisioned snub-nosed monkeys in the Baima Snow Mountain National Nature Reserve in Yunnan, we explored the effect of food-provisioning on gut flora of the monkeys. [Results] Food-provisioning significantly increased the richness, evenness, and phylogenetic diversity of the gut flora and reduced the variance of community composition among monkey individuals. linear discriminant analysis effect size (LEfSe) analysis revealed that food-provisioning significantly changed the relative abundance of 20 bacterial taxa. To be specific, it increased the abundance of 8 taxa, such as Firmicutes, and decreased the abundance of 12 taxa, including Proteobacteria and Bacteroidetes. The correlation network analysis revealed that wild monkey group had more complex and robust microbial correlation network. Kyoto encyclopedia of genes and genomes (KEGG) functional prediction found that food-provisioning mainly reduced the degradation ability as it added highly degradable nutrients. [Conclusion] Food-provisioning influences multiple aspects of gut flora of Yunnan snub-nosed monkeys, particularly the biodiversity, community composition, and correlation network.

Abstract:[Background] Qiwei Baizhu San can be used to treat diabetes caused by deficiency of both qi and yin. Especially in recent years, it has been widely used in improving insulin resistance of diabetes mellitus. [Objective] To observe the effects of Qiwei Baizhu San on intestinal barrier and inflammatory factors in diabetic rats and to explore its mechanism. [Methods] Sixty SD male rats in specific pathogen free (SPF) grade were randomly divided into a normal control group, a model group, a metformin group, and high, medium, and low-dose Qiwei Baizhu San groups, with 10 rats in each group. The diabetic rat model was established by intraperitoneal injection of streptozotocin (STZ). Rats in the normal control group were given a normal diet, and rats in the model group, the metformin group, and the high, medium, and low-dose Qiwei Baizhu San groups were all given high-sugar and high-fat diets. After successful modeling, the metformin group and the Qiwei Baizhu San groups were treated with metformin and Qiwei Baizhu San by gavage, respectively. Normal saline was given by gavage in the normal control group and the model group. The general living status of rats before and after treatment was observed. After 4 weeks of drug intervention, fasting blood glucose (FBG), fasting insulin (FINS), and lipopolysaccharide (LPS) were detected by an automatic biochemical analyzer. Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in rat serum were measured by enzyme-linked immunosorbent assay (ELISA). Hematoxylin-eosin (HE) staining was used to observe the pathological changes in colon tissues of rats in each group, and immunohistochemistry was used to observe the expression position and level of occludin protein in colon tissues. Western blotting assay was used to determine the protein expressions of zonula occludens-1 (ZO-1), occludin, toll-like receptor 4 (TLR4), and nuclear factor kappa B (NF-κ B). [Results] As compared with the normal control group, the FBG of rats after modeling in each group was ≥11.1 mmol/L (P <0.01). As compared with the model group, the FBG of rats in the low and medium-dose Qiwei Baizhu San groups decreased, but the difference was statistically insignificant (P >0.05). The FBG of rats in the high-dose Qiwei Baizhu San group and the metformin group decreased as compared with the model group (P <0.05). As compared with the model group, the levels of FINS, LPS, IL-6, and TNF-α in the metformin group and high, medium, and low-dose Qiwei Baizhu San groups decreased (P <0.01). As compared with the blank group, the intestinal cells in the model group were disorderly arranged, the intestinal villi were incompletely broken and sparsely spaced, the goblet cells were significantly reduced and unevenly distributed, accompanied by neutrophil exudation, and the average optical density of occludin protein was reduced (P <0.01). As compared with the model group, the intestinal villi of the rats in the high-dose Qiwei Baizhu San group and the metformin group were more arranged neatly and completely, the number of colonic goblet cells was restored to varying degrees, the neutrophil exudation was reduced, and the average optical density of occludin protein was increased to varying degrees (P <0.01). The results of Western blot showed that as compared with the model group, the protein expressions of ZO-1 and occludin in the ileum tissues of rats in the high-dose Qiwei Baizhu San group and the metformin group increased (P <0.01), and the protein expressions of toll-like receptor 4 (TLR4) and nuclear factor kappa B (NF-κ B) decreased (P <0.01). [Conclusion] Qiwei Baizhu San can up-regulate the expression of tight junction protein in the intestine, promote the repair of intestinal mucosal mechanical barrier, maintain the integrity of tight junctions, reduce the level of serum LPS, inhibit the TLR4/NF-κ B pathway, and reduce the release of IL-6, TNF-α, and other inflammatory factors, thus reducing the inflammatory reaction and improving insulin resistance of the body.

Abstract:Wolbachia is a common group of intracellular bacteria in arthropods. They can alter host biology in diverse ways, including the induction of reproductive manipulations, such as feminization, thelytoky, male killing, and cytoplasmic incompatibility (CI). Among them, CI, the most common phenotype, refers to the fact that embryonic lethality occurs when a Wolbachia -infected male insect mates with a Wolbachia -uninfected or incompatible female insect, whereas the cross between the male and female both infected with the same strain of Wolbachia is compatible. CI is caused by linked pairs of Wolbachia genes known as CI factors (CifA and CifB). CifB as a toxin can induce CI in male insects, while CifA as an antidote can rescue CI in female insects. This review summarized the research on the structure, function, and mechanism of CI factors, aiming to provide a solid base for the future application of Wolbachia and CI in the prevention and control of mosquito-borne diseases and agricultural pests.

Abstract:Periodontal disease is a chronic infectious disease involving periodontal supporting tissue. Microorganisms and their metabolites in dental plaque biofilm are key initiating factors of periodontal disease, which can lead to oral microecological imbalance and host immune response. At present, periodontal disease is mainly treated by mechanical removal of dental plaque biofilm and calculus, which has limited therapeutic effect. Probiotics can improve the balance of oral microecology and promote the treatment of periodontal diseases by producing antibacterial substances, stimulating local immune responses, and competing with pathogenic bacteria for mucosal receptors and nutrients. This paper reviews the experimental and clinical studies, mechanism of action, and safety of probiotics in the treatment of periodontal disease in recent years, providing reference for the application of probiotics in this field in the future.

Abstract:Natural products, which feature complex structures and diverse activities, are important sources of new drugs. The research on the biosynthesis pathways of natural products is conducive to exploring the synthesis mechanism catalyzed by enzymes and promoting the application of complex natural products. The corresponding gene clusters regulate the biosynthesis of natural products, and a large number of biosynthetic gene clusters (BGCs) of natural products are not expressed or expressed at low levels in wild-type strains. Thus, these gene clusters are usually cloned before further research on them. It remains challenging to clone and express large gene clusters for the discovery of novel natural products. Construction of genomic library, transformation-associated recombination (TAR), and Red/ET recombination technology are essential techniques for cloning large gene clusters. This study summarizes the research on these three cloning techniques from the aspect of cloning strategy and application. Moreover, we discuss the challenges in cloning large gene clusters, providing a methodological reference for further study.

Abstract:The human skin surface is home to a variety of microorganisms that are closely associated with skin health. Staphylococcus epidermidis is one of the major microorganisms on the skin surface of the normal population and plays a role in maintaining skin health. Under normal physiological conditions, S. epidermidis participates in innate immunity of skin through antimicrobial peptides, and its cell wall component, lipoteichoic acid, contributes to the development and initiation of the adaptive immune system, thereby regulating the skin immune process. By secreting sphingomyelinase, S. epidermidis can replenish ceramide in the skin and enhance the tight junctions of keratinocytes, thus maintaining skin barrier homeostasis. S. epidermidis can communicate with a wide range of bacteria and plays a benign role in the antimicrobial defense of the skin. Furthermore, S. epidermidis promotes skin re-epithelialization to accelerate wound repair. This review summarizes the latest findings about the role of S. epidermidis in the maintenance of skin health, contributing to the in-depth understanding of its beneficial effects on the skin as a potential probiotic to be fully utilized for the treatment of dermatological diseases and the development of cosmetics.

Abstract:The rumen of ruminants is known as a natural fermenter with the strongest fiber-degrading ability. The various microorganisms inhabiting the rumen confer it the ability to digest plant feed. As a group of microorganisms with low abundance in the rumen, anaerobic fungi are the first to colonize the cellulosic feed ingested by host animals and degrade lignocellulose by secreting massive efficient carbohydrate-active enzymes. However, due to the lack of sufficient genomic information and an effective genetic operating system for anaerobic fungi, the research on fiber-degrading enzymes secreted by anaerobic fungi and their degradation mechanisms is limited. This paper summarized the taxonomy and published genome information of the anaerobic fungi in the rumen, introduced the fiber-degrading enzymes and the structure and catalytic mechanism of cellulosomes, and recapitulated the applications of such enzymes in biomass energy, feed processing, textile, papermaking, and food processing. Studying the properties of fiber-degrading enzymes in anaerobic fungi will improve our understanding of their ability to degrade lignocellulose and their competition for resources in a highly complex rumen environment. Further, it will help us to learn the application potential of the enzymes in biotechnology and provide new strategies for the application of the enzymes in industries.

Abstract:Rice feeds nearly half of the world’s population, and it is also considered as the model host plant for studying the symbiosis between arbuscular mycorrhizal fungi (AMF) and non-legumes. The diversity and ecological roles of AMF in paddy fields have not been well understood for a long time due to the limitation of research methods. Studies have demonstrated that AMF are ubiquitous in paddy fields with the development of high-throughput sequencing technology in the last decade. The application of new techniques greatly promoted the research on the ecological roles of AMF and the interactions between AMF and rice in paddy ecosystems. In this review, we summarized the process of the establishment of arbuscular mycorrhizal (AM) symbionts, the diversity and influencing factors of AMF, and the ecological roles of AMF in paddy ecosystems. Furthermore, the potential application of AMF in rice cultivation and future research on the interactions between AMF and rice have been prospected. The establishment of AM symbionts depends on a series of complex signal recognition, exchange, and conduction between AMF and rice. The AMF colonization rate of rice plants is higher in the upland environment than that in the paddy environment, and it is affected by host plants, environmental factors, and cultivation management measures. AMF play a key role in regulating rice growth and nutrient absorption, resisting environmental stress, and reducing methane (CH₄) and nitrous oxide (N₂O) emissions from paddy fields. AMF can act together with other microorganisms to help rice absorb nutrients and resist environmental stresses. This paper reviews the research progress and development trends of AMF in paddy fields and discusses the inadequacy in this research field. In the future, efforts should be made to explore the relationship between the diversity and ecological roles of AMF, the mutual signal recognition between rice and AMF, and the molecular cascade reactions in the establishment and maintenance of AM symbiosis. Moreover, it is essential to understand the underlying molecular mechanisms of AMF promoting rice nutrient absorption, enhancing rice stress resistance, and reducing greenhouse gas emissions from rice fields.

Abstract:The changes of natural conditions and human activities have not only intensified soil acidification and expanded the area of acidic soil but also seriously affected soil nitrogen cycle. As a rate-limiting step of nitrification, ammonia oxidation is the core of the global nitrogen cycle, which has attracted extensive attention. Exploring ammonia oxidation in acidic soil and the involved functional microorganisms is of great significance for deciphering the nitrogen cycle mechanism and promoting soil nutrient cycling. This paper summarized the mechanisms of ammonia oxidation in soil and compared the relative contributions of ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA), and complete ammonia oxidizers (Comammox) to ammonia oxidation in acidic soil. Then, we analyzed the effects of microbial endogenous function differences and environmental factors such as pH and substrate concentration on the abundance, activity, and community structure of ammonia-oxidizing microorganisms. Finally, the future studies about ammonia-oxidizing microorganisms were prospected. This paper is expected to provide scientific references for future research on ammonia oxidation in acidic soil, as well as the application and practice of microbial remediation.

Abstract:Microbiology is an important professional course for the aquaculture major. The experiment teaching of Microbiology is a key approach to convert theoretical knowledge into practical ability, which plays an essential role in the training of professional personnel. However, the operation of microbiological experiment is complicated and time-consuming. In the limited experimental teaching time, students cannot systematically participate in the complete experimental process, which results in the unsatisfactory teaching effect. We have explored the mode of undergraduate assistant in the teaching of Microbiology Experiment. The teaching assistants used their fragmented time to participate in experimental design, preparation of reagents and media, isolation and purification of microorganisms and mastered strong experimental skills. Then, they went back to the classroom to act as a bridge between teachers and students, assisting teachers to guide students’experiments. This mode enhanced the learning interest and comprehensive quality of the assistants, corrected the students’mistakes during the experiment in time, and improved the teaching effect.