Abstract:[Background] Low level expression and poor secretion efficiency are the common problems in the production of secretory recombinant proteins by industrial yeast. [Objective] To investigate the effect of inactivating yapsin proteases Yps1p and Yps2p on β-glucosidase expression in Saccharomyces cerevisiae An-α, a yeast strain isolated from Angel industrial yeasts. [Methods] By utilizing CRISPR/Cas9-based gene editing technology, we firstly constructed a UPR-indicator strain An-α(leu2::UPRE-lacZ), designated as An-αL. Next, the YPS1 and YPS2 genes were inactivated, followed by transformation of the β-glucosidase-expressing plasmid BG that was constructed on the YEplac195 vector. The growth and enzymatic activities of the resultant strains were evaluated. [Results] Inactivation of the YPS1 or YPS2 gene of the strain An-αL did not affect the cell growth in YPD medium. The presence of the plasmid BG increased maximum OD₆₀₀ values in the YPC medium by 21.9% and 7.4%, respectively. The maximum enzymatic activities were 0.087 5 and 0.068 6 U/(mL·OD₆₀₀), which were 2.268 and 1.778 times of the control value, respectively. The ratio of the secreted protein was also increased by 19.4% and 22.2%, respectively. There was a good correlation between the β-glucosidase activity level and the UPR signal response value as represented by the β-galactosidase activity level. [Conclusion] Inactivation of YPS1 and YPS2 could improve the secretory β-glucosidase yield of the strain An-α.

Abstract:[Background] Acetyl-CoA acetyltransferase (Acat) belongs to thiolase family and it can be classified into two types (I and II). Type II Acat acts as the first rate-limiting enzyme of the mevalonate (MVA) pathway, and its expression level and catalytic activity affect the production of terpenoids and their derivatives. [Objective] To study whether Acat2 overexpression can increase the carotenoid biosynthesis in Rhodosporidium kratochvilovae. [Methods] A gene encoding type II Acat, named RKAcat2, was cloned from R. kratochvilovae strain YM25235 and then introduced into YM25235 for overexpression. [Results] Compared with the control, RKAcat2 overexpression in YM25235 increased the carotenoid content by 50.53% while decreased the lipid content, oleic acid content, and acetyl-CoA level by 22.80%, 17.78%, and 13.64%, respectively. [Conclusion] RKAcat2 overexpression promoted the entry of more acetyl-CoA into MVA pathway. Consequently, the carotenoid biosynthesis in YM25235 was increased, which is consistent with the results of transcriptional analysis of some genes in MVA and carotenoid biosynthesis pathways. This study may provide a reference for further research on improving the contents of carotenoid and its specific compositions in oil-producing red yeast by metabolic engineering.

Abstract:[Background] Bacteria can synthesize medium-chain-length polyhydroxyalkanoates (MCL-PHA) through fatty acid metabolic pathway coupled with PHA synthetic pathway. MCL-PHA’s tensile strength, glass transition temperature, and other processing properties better than the short-chain-length PHA, are the promising candidate for PHA industrialization. [Objective] In order to obtain new PHA-producing bacteria that can metabolize oils, we used three methods to isolate bacteria from marine sediments, identified them, and then evaluated the PHA-producing ability. [Methods] We collected the offshore sediment sample of Dapeng Bay in Shenzhen, and isolated bacteria directly from the sample, each week during the 5-week incubation, or during the 10-week enrichment with stepwise increase of oil content (1%–10%) and salt content (3.5%–12.5%). Then, we identified the strains based on 16S rRNA gene similarity and phylogenetic analysis, tested the PHA-producing ability by sequencing the gene of PHA synthase (PhaC), and determined the types, metabolic pathways, and phylogenetic relationship of PhaC according to the draft genome. The content and composition of PHA were detected by gas chromatography. [Results] A total of 96 strains were isolated and 38% had the phaC gene. Among them, 9 genera had not been reported to produce PHA: Acuticoccus, Idiomarina, Halobacillus, Microbulbifer, Maritimibacter, Nitratireductor, Pelagibaca, Pseudooceanicola and Thalassospira, and 8 of the 9 genera except Microbulbifer were enriched with oil. Thus, the enrichment with oil helped to discover new PHA-producing bacterial resources. In addition, we obtained 2 strains with high intracellular PHA content: N. aquimarinus SY-2-4 could accumulate PHA of 35.0% dry cell weight when cultured in nutrient broth, while Roseibium aggregate SN13-21 could use pyruvate as sole carbon source to accumulate PHA of 19.9% dry cell weight. [Conclusion] The offshore environment of Dapeng Bay harbors rich PHA-producing bacterial resources that are worthy of further investigation. The enrichment method of stepwise increase of oil content and salt content is beneficial to isolating different PHA-producing bacteria. Next, fermentation optimization of the 2 high-PHA-content strains will be further investigated.

Abstract:[Background] Some dissimilatory Fe(III)-reducing bacteria have the ability of dissimilatory iron reduction and fermentative hydrogen production, which can provide an important way to study the mechanism of fermentative Fe(III)-reducing bacteria. [Objective] This study aimed to screen a fermentative Fe(III)-reducing bacterium with the characteristics of microbial Fe(III) reduction and hydrogen production. [Methods] The strain was screened by three-layer plate method and identified by sequencing the 16S rRNA gene. Fe(II) concentration and hydrogen production were investigated to analyze the characteristics of microbial Fe(III)-reducing and hydrogen-producing by this strain. [Results] Compared to Clostridium butyricum, the similarity was 100% by strain LQ25 based on 16S rRNA gene sequence. The results show that cell growth by strain LQ25 under Fe(III)-reducing conditions using Fe(OH)₃ as electron acceptor were significantly enhanced compared with the control (no adding Fe(III)). Strain LQ25 was able to grow, reduce Fe(III) and produce hydrogen when using pyruvate, glucose and lactate as electron donor respectively. The highest Fe(II) concentration and cell growth by strain LQ25 was achieved when pyruvate was used as electron donor, with protein content (78.88±3.40) mg/L and Fe(II) concentration (8.27±0.23) mg/L. When glucose was used as electron donor, hydrogen production was (475.2±14.4) mL/L under Fe(III)-reducing conditions with the adding Fe(III), which was 87.7% higher compared to the control (no adding Fe(III)). [Conclusion] A fermentative Fe(III)-reducing bacterium Clostridium sp. LQ25 which is capable of microbial Fe(III) reduction and hydrogen production was successfully screened in this study. The result will provide new experimental evidence for the mechanism of extracellular electron transfer by fermentative Fe(III)-reducing bacteria.

Abstract:[Background] Fungi and bacteria are considered to play a synergistic role in the bioremediation of polycyclic aromatic hydrocarbons (PAHs)-contaminated soil. However, there are limited studies on their synergistic degradation in soil. [Objective] This paper aims to investigate the different PAHs-degrading abilities of Pleurotus ostreatus and Sphingobium sp. NS7 and reveal their synergistic role in the biodegradation of anthracene and benzo[a]anthracene in soil. [Methods] The PAH degradation by P. ostreatus and Sphingobium sp. NS7 was performed in liquid culture. Soil microcosms were set up with radiolabeled anthracene and benzo[a]anthracene to investigate the fate of PAHs after bioaugmentation. [Results] NS7 can efficiently degrade low-molecular-weight (LMW) PAHs, as it removed almost all the anthracene when anthracene was the only carbon source. In the case of compound contamination, the bacterium removed >90% of phenanthrene, anthracene, fluoranthene, and pyrene while degraded benzo[a]pyrene with a low rate (9.76%). By contrast, P. ostreatus demonstrated relatively high removal rate (21.18%) of benzo[a]pyrene. The degradation rate of LMW PAHs by P. ostreatus was much lower than that by NS7. In the natural soil, the mineralization rate of anthracene and benzo[a]anthracene was 18.61% and 4.28%, respectively. P. ostreatus promoted benzo[a]anthracene mineralization in natural soil (increased by 2.24 folds) but NS7 failed to significantly improve anthracene mineralization. Thus, the competition of indigenous microbial communities might affect the survival of exogenous microorganisms. A sterile soil system excluding the microbial competition was used to investigate the helper role of fungal hyphae in the synergistic degradation. With the help of fungal hyphae, the mineralization of anthracene was enhanced by NS7 (mineralization rate up from 1.75% to 5.91%), but no promotion effect was observed in benzo[a]anthracene mineralization. Therefore, the reason for the enhancement of benzo[a]anthracene mineralization in natural soil by P. ostreatus might be that the fungal hyphae promoted the migration of indigenous PAHs-degrading bacteria in soil matrix and thus enhanced the contact of them with the contaminant. [Conclusion] Bacteria can efficiently degrade LMW PAHs while fungi showed high degradation rate of high-molecular-weight PAHs like benzo[a]pyrene. Enhanced PAH mineralization in natural soil was observed after P. ostreatus inoculation, and the mechanism might be that the fungal hyphae promoted migration of indigenous bacteria in the soil matrix. This study deepens our understanding of the synergistic degradation of PAHs in soil by fungus and bacterium and lays a theoretical basis for soil bioremediation based on the synergistic effect of fungi and bacteria.

Abstract:[Background] China is a major agricultural producer, with a large proportion of fishery, forestry, agriculture, and animal husbandry. Organic pesticides are widely used in animal husbandry and aquaculture, particularly the organophosphorus pesticides (OPs) which feature low toxicity and little residue. OP indirectly affects human health through biological enrichment, and the resulting reproductive toxicity cannot be ignored. Photosynthetic bacteria are environmentally friendly beneficial bacteria in water, some of which can degrade organic pesticides. [Objective] We isolated a photosynthetic strain (No. SPZ) from the Minghu Lake of Shanghai Ocean University and tested its tolerance to and degradation of phoxim, aiming at provide a strain for the removal of OPs in aquaculture water. [Methods] The strains used in this study were identified by 16S rRNA gene sequencing. The OD₆₆₀ of the isolated strain SPZ and the standard strain ST at different inoculum sizes was measured by UV spectrophotometry to determine the optimal inoculum size. The toxicity of phoxim to photosynthetic bacteria was determined based on the changing trends of OD₆₆₀ of the bacteria exposed to phoxim at different concentration levels. The phoxim-degrading ability of SPZ and ST was determined by high performance liquid chromatography (HPLC), and that of live and dead (from heating) SPZ was also detected (HPLC) to clarify the degradation mechanism. [Results] The 16S rRNA gene sequence analysis demonstrated that SPZ showed 99% similarity to Rhodopseudomonas and clustered with Rhodopseudomonas palustris ATCC 17001 (confidence: 100%). The optimal inoculation volume fractions of SPZ and ST were 10%. SPZ can tolerate 100 mg/L phoxim, and phoxim significantly inhibited the growth of the photosynthetic bacteria at the concentration > 100 mg/L. Phoxim was added to photosynthetic bacteria at logarithmic growth phase and the relative degradation of phoxim was 12.97%-26.69% (at 20.0 mg/L), 24.25%-32.85% (at 2.0 mg/L), and 16.66%-34.59% (at 0.2 mg/L) after 1 day, 46.63%-53.95% (at 20.0 mg/L), 24.78%-30.34% (at 2.0 mg/L), and 31.92%-39.25% (at 0.2 mg/L) after 3 days, and 93.65%-97.72% (at 20.0 mg/L), 67.69%-74.41% (at 2.0 mg/L), and 10.34%-24.27% (at 0.2 mg/L) after 5 days. [Conclusion] The common photosynthetic SPZ can effectively remove the phoxim in water. It has a broad application prospect in aquaculture and OPs-contaminated wastewater treatment.

Abstract:[Background] Isaria farinosa with global distribution and wide host range is one of common entomopathogenic fungi in natural environment. However, the morphological characteristics of many species of Isaria are similar, which are easy to be confused only according to the classical morphological identifications. For a long time, more and more controversies and confusions have emerged on the accurate classification status of the strains identified as I. farinosa according to their morphological characteristics. Recently, its phylogeny has proved multigenus. [Objective] To clarify the taxonomic position of I. farinosa and explore the natural taxonomic status of the strains which were previously identified as I. farinosa. [Methods] Six strains previously identified as I. farinosa were selected for phylogenetic analysis of five gene loci (nrSSU, nrLSU, TEF, RPB1 and RPB2) and morphological methods to reconfirmed their taxonomic status. [Results] Through checking on the literature and experimental research, the phylogeny of I. farinosa was clarified. The results showed that all the tested strains from different sources were far related to the model strain of I. farinosa, but they are identical to a new combination species Samsoniella hepiali. [Conclusion] In this study, the correct taxonomic status of 6 Isaria-like strains was revised as S. hepiali by using the classical morphological characteristics and constructing the five genes phylogenetic tree. To provides a reasonable method for the correct identification of Isaria-like fungi and scientific guidance for further utilization of these strains.

Abstract:[Background] The fast-growing Gram-negative bacterium Vibrio natriegens is a burgeoning tool in biotechnology. Previous research has mainly focused on developing tools for in vitro and in vivo recombinant protein production using V. natriegens. However, many physiological activities that support fast growth and protein production remain largely uncharacterized. Ubiquitously produced by bacteria, outer membrane vesicles (OMVs) not only carry out important functions but also can serve as a useful delivery tool for vaccine and therapeutics development. [Objective] Characterize the proteomes of OMVs during exponential phase growth and to employ OMVs for heterologous protein delivery. [Methods] Using transmission electron microscopy, dynamic light scattering, and mass spectrometry, we characterized the morphology and size distribution of extracted OMVs and their protein composition. We used the superfolded green fluorescent protein (sfGFP) as cargo to determine OMVs protein carriers. [Results] OMVs of mid- and late-exponential phases cultures contain 288 and 317 proteins, respectively. These proteins belong to multiple functional groups including ABC transporters, flagella and two-component systems. By contrast, we identified 1 480 and 1 565 proteins in whole cell samples under these two conditions, respectively. We screened OMV proteins for candidate carriers and found an OmpA-family protein that we name OmpA24 could enrich the sfGFP as a protein-fusion cargo in OMVs. [Conclusion] We demonstrate for the first time that V. natriegens can produce OMVs throughout exponential growth and present the first proteomic snapshot of OMVs and related whole cell samples under different growth phases. OmpA24 protein is a promising carrier for delivery of heterologous protein-fusion cargo into OMVs. This study will facilitate the application of V. natriegens in protein expression and OMV-mediated secretion.

Abstract:[Background] β-glucosidase is an important class of cellulolytic enzymes. At present, β-glucosidase (Bgl) derived from cultivable microorganisms has the problems of poor thermal stability and acid-base stability and a narrow range of action. [Objective] Discover new β-glucoside genes from the fecal microbial metagenomics of Rhinopithecus bieti, express heterologously and study its enzymatic properties, and provide new enzyme resources for food and other fields. [Methods] Starting from the fecal microbial metagenome, the β-glucosidase genes BglRBS_26 and BglRBS_9 were amplified and heterologously expressed, and the enzymatic properties were studied. [Results] The GH1 family recombinant β-glucosidase BglRBS_26 and BglRBS_9 were obtained, with molecular weights of 60 kD and 50 kD, respectively. The optimal conditions for BglRBS_26 are pH 6.0 and 45 ℃; the optimal conditions for BglRBS_9 are pH 5.0 and 40 ℃. The K_m of BglRBS_26 and BglRBS_9 are (0.681 6±0.164 2) μmol/L and (3.317 0±0.871 4) μmol/L, respectively. BglRBS_26 has good acid-base tolerance. After treatment at pH 5.0–6.0 for 1 h, the remaining enzyme activity is greater than 110%; in the range of pH 7.0–8.0, the remaining enzyme activity remains above 100%. In addition, sucrose can activate BglRBS_26 and BglRBS_9 to varying degrees. When 20% (M/V) sucrose is added to the reaction system, the enzyme activity of BglRBS_26 can be increased to 140%; 10% (M/V) sucrose can increase the enzyme activity of BglRBS_9 to 180%. In addition, BglRBS_26 has better NaCl tolerance and stability. It retains 80% enzyme activity after being treated at 37 ℃ and 2.5 mol/L NaCl for 1 h. [Conclusion] In this study, two novel β-glucosidase genes BglRBS_26 and BglRBS_9 were obtained from the R. bieti fecal microbial metagenome, and they were successfully expressed in E. coli BL21(DE3). BglRBS_26 and BglRBS_9 have good pH stability and sucrose tolerance, making them have potential applications in food, fermentation and other industries.

Abstract:[Background] Mortierella alpina is an oleaginous fungus that can accumulate a large amount of arachidonic acid (AA). The fatty acids produced by M. alpina are mainly assembled on the glycerol skeleton and present in the form of triacylglycerol (TAG). Diacylglycerol acyltransferase (DGAT) is a key enzyme in the biosynthetic pathway of TAG and plays an important role in TAG production of M. alpina. [Objective] This study explored the functional characteristics of M. alpina DGAT2 in TAG biosynthesis, aiming to provide a reference for promoting the TAG production of oleaginous fungi and improving the fatty acid composition of TAG. [Methods] Two candidate genes MaDGAT2A/2B were screened from the M. alpina ATCC32222 genome by sequence alignment. Functional characterization of MaDGAT2A/2B were performed by heterologous expression in Saccharomyces cerevisiae. Then the activity of MaDGAT2A/2B was further analyzed by detecting TAG yield under the condition of exogenous addition of AA. Finally, the in vivo activity of MaDGAT2A/2B was analyzed by detecting the total fatty acid yield and composition of the M. alpina recombinant strains overexpressing MaDGAT2A/2B. [Results] The transforming with MaDGAT2A gene could increase the production of TAG in S. cerevisiae to 3.06% of dry cell weight, which was 4.91-fold of the control group; while MaDGAT2B did not significantly increase the yield of TAG in S. cerevisiae. When AA was added exogenously, MaDGAT2A/2B could promote TAG synthesis in the yeast recombinant strains. The TAG content of recombinant yeast expressing MaDGAT2A was 3.67-fold of the control group. The TAG content of recombinant yeast expressing MaDGAT2B was 2.61-fold of the control group. Overexpression of MaDGAT2A/2B in M. alpina had no significant effect on the total fatty acid yield, but could significantly increase the content of AA in total fatty acids, up to 39.15%, an increase of 16.14% compared to the control group. [Conclusion] MaDGAT2A/2B could participate in the biosynthesis of TAG, indicating that the two proteins encoded by the two candidate genes have DGAT activity. And MaDGAT2A/2B could increase the content of AA in M. alpina, which is of great significance for improving the lipid composition of oleaginous fungi and increasing their application value.

Abstract:[Background] Wheat/maize rotation is one of the main cropping patterns of grain crops in China. At present, there is still a lack of comprehensive understanding of the microbial diversity in rhizosphere soil of wheat/maize rotation field. [Objective] The objective of this thesis is to identify the variation of wheat/maize rhizosphere soil microorganisms and understand their potential functions. [Methods] In this study, wheat/maize rhizosphere soil was used as material, and bacterial 16S rRNA gene and fungal rDNA ITS gene sequencing were used to analyze the microbial diversity of wheat/maize rhizosphere soil. [Results] The results showed that the abundance of microorganisms in maize season was higher than that in wheat season, but there was no significant difference in diversity. Actinobacteria, Proteobacteria, Acidobacteria and Chloroflexi were the dominant phyla of bacterium in the rhizosphere soil in wheat and maize seasons, while Ascomycota was the dominant phylum of fungi. There were 631 and 261 common bacteria and fungus in wheat and maize seasons. There were 38 and 58 unique bacteria and fungus in wheat season, and 25 and 39 unique bacteria and fungus in maize season, respectively. LEfSe analysis (LDA threshold is 2) of bacterium and fungi showed that Actinobacteria and Microascules were enriched in wheat season. Meanwhile, Sphingomonadales and Tremellomycetes were enriched in maize season. Compared with wheat season, the abundance of metabolic pathways involved in nutrient cycling in maize season was higher, while the abundance of metabolic pathways involved in oxidative stress was lower. [Conclusion] The results have theoretical and practical significance for guiding the management of wheat/maize rotation fields.

Abstract:[Background] Sclerotinia disease is one of the main root diseases of Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim) Kitag. Trichoderma is extensively used for biocontrol owing to the fungistatic activity. Recently, the control of Sclerotinia asari by Trichoderma has attracted the interest of scholars. [Objective] Trichoderma strains were isolated from the rhizosphere soil of healthy A. heterotropoides Fr. Schmidt var. mandshuricum (Maxim) Kitag. with the dilution-plate method and those antagonistic to S. asari were screened out. [Methods] The inhibition of the isolated strains (plate confrontation method), the volatile and non-volatile strain metabolites, and strain fermentation broth (growth rate method) on S. asari was respectively determined. The malondialdehyde (MDA) content (thiobarbituric acid method), catalase (CAT) activity (ultraviolet absorption method), superoxide dismutase (SOD) activity (nitroblue tetrazolium assay), and peroxidase (POD) activity (guaiacol method) of S. asari treated with the selected Trichoderma were determined. [Results] A total of 14 Trichoderma strains were isolated and identified based on morphological observation and ITS-RPB2 sequence alignment as T. harzianum, T. hamatum, T. koningiopsis, T. atroviride, T. brevicompactum, and T. tomentosum, respectively. The inhibition rates of T. hamatum A26, T. koningiopsis B30, T. hamatum C6, and T. harzianum A17 on S. asari were all above 90%. The volatile metabolites of T. hamatum C6 demonstrated the highest inhibition rate (53.73%±0.07%), and the non-volatile metabolites showed stronger inhibitory effect, particularly those of A17 (inhibition rate: >75%), A26 (inhibition rate: >75%), C6 (inhibition rate: >75%), and B30 (inhibition rate: 100%). Thus, strains A17, A26, B30, and C6 had the strongest control effect and the inhibition rates of the fermentation broth of these 4 Trichoderma strains against S. asari were 56.33%±0.12%, 77.22%±0.06%, 82.28%±0.03%, and 46.20%±0.04%, respectively. After being treated with the non-volatile metabolites of the 4 strains for 7 days, S. asari saw significantly increased MDA content, particularly the S. asari strains treated with the non-volatile metabolites of A26 (MDA content was 7.7 times that of the control). Besides, the antioxidant enzyme activity of S. asari decreased. To be specific, the activity of CAT, SOD, and POD declined by 19.67%-75.84%, 4.71%-68.71%, and 3.57%-67.86%, respectively, as compared with that of the control. [Conclusion] Trichoderma A17, A26, B30, and C6 isolated from the rhizosphere soil of healthy plants of A. heterotropoides Fr. Schmidt var. mandshuricum (Maxim) Kitag. can be used for the biocontrol of S. asari.

Abstract:[Background] Tobacco black shank is a fungal disease caused by Phytophthora parasitica var. nicotianae, which has brought great losses to tobacco production in China. [Objective] In this study, a strain MC4-2 was isolated from Periplaneta americana intestinal tract, which had antagonistic effect on P. parasitica var. nicotianae. [Methods] The strain MC4-2 was identified by morphological characteristics, physiological and biochemical methods and 16S rRNA gene sequencing. The fermentation medium and fermentation conditions of strain MC4-2 were optimized by single factor and orthogonal experiment using the OD value of bacterial fermentation broth at the wavelength of 600 nm as the index. The control effect of this strain on tobacco black shank was confirmed by potted experiment in greenhouse. [Results] Through the plate confrontation test, it was found that strain MC4-2 had a good inhibitory effect on P. parasitica var. nicotianae, and the inhibition rate could reach 64.04%; 16S rRNA gene sequence analysis showed that the similarity between strain MC4-2 and Bacillus subtilis reached 99%, and the morphological and physiological and biochemical characteristics were basically consistent with that of strain MC4-2; The optimal medium formula was peptone 1.0 g, yeast extract 1.0 g, sucrose 1.5 g, distilled water 100 mL. The results showed that the optimal fermentation conditions were inoculation amount of 8%, liquid volume of 30 mL, initial pH of 6.0, rotation speed of 210 r/min, temperature of 32 ℃, culture time of 60 h and light time of 12 h/d. The results of indoor pot experiment showed that the average control effect of strain MC4-2 on tobacco black shank could reach 63.86%. [Conclusion] This study provides an important reference for the biological control of B. subtilis MC4-2.

Abstract:[Background] Studies have shown that rhizosphere microorganisms play an important role in the root ecosystem, influencing the nutrient absorption and health of plant. [Objective] To understand the culturable microbial diversity in the rhizosphere of flue-cured tobacco variety K326 growing in four tobacco fields, which does not occur disease for many years, and to screen strains with the biocontrol and plant-growth promoting function for tobacco diseases control. [Methods] Traditional culture-dependent methods were used to isolate and identify bacteria and fungi in tobacco rhizosphere soil. The plant growth-promoting characteristics and pathogen antagonism of the strains were evaluated, and the growth-promoting effects of typical strains were further verified on tobacco seedlings. [Results] In this study, 261 isolates were obtained, including 160 bacterial strains and 101 fungal strains. Bacterial phyla of Proteobacteria and Firmicutes and fungal phyla of Ascomycota and Mucoromycota are the main groups. At the genus level, Pseudomonas and Bacillus were the most abundant genera in bacteria, and Aspergillus and Penicillium dominated in fungi. Next, 44 bacterial strains were selected from different species as representatives. We found that they all have IAA production ability, 9 strains can dissolve organic phosphorus, 16 strains can dissolve inorganic phosphorus, 13 strains produce siderophores, and 14 strains produce ACC deaminase. 25 and 26 strains that inhibiting the pathogens of bacterial wilt and black shank diseases were obtained from 160 bacterial strains, respectively. The pot experiment found that P. koreensis HCH2-3, P. lurida FGD5-2 and B. velezensis EM-1 can promote growth of tobacco seedlings, and consortium of these three strains has the most obvious growth-promoting effect. [Conclusion] There are numerous microorganisms with the potential to control disease and promote plant growth in the tobacco rhizosphere. Application of synthetic bacteria or functional complementary strains is an important direction for the development of microbial agents in the future.

Abstract:[Background] Calcium/calmodulin-dependent protein kinase (CaMK), an important downstream target protein of calmodulin in the calcium signaling pathway of eukaryotic cells, plays an important role in pathogen growth, stress response and pathogenicity. [Objective] Cloning, bioinformatics and expression analysis of AaCaMK gene on infection structure differentiation of Alternaria alternate, casual agent of pear black spot, for further clarifying the molecular regulatory role of the AaCaMK gene in the calcium signal pathway on the infection structure differentiation of A. alternata. [Methods] The AaCaMK gene was cloned from Alternaria alternata JT-03 by homologous cloning; The AaCaMK gene was analyzed by TMHMM, ProtScale, SOPMA and other software; Real-time quantitative PCR (RT-qPCR) was used to analysis of the expression of AaCaMK on infection structure differentiation of A. alternata. [Results] Three isotypes of calcium/calmodulin-dependent protein kinase designated AaCaMK1, AaCaMK2 and AaCaMK3 were identified in A. alternata with length of 1 212, 1 200 and 2 349 bp; The bioinformatics analysis revealed that these three CaMK all contain the PKC_like superfamily domains (PKC_Like Superfamily). The conserved kinase domains of both AaCaMK1 and AaCaMK2 belonged to the catalytic domain of CaMK ser/thr protein kinase (STKc_CaMK) while that of AaCaMK3 belonged to the catalytic domain of liver kinase B1 (LKB1) and calmodulin dependent protein kinase kinase (CaMKK)(STKc_LKB1_CaMKK); The homology analysis showed that the homology of AaCaMK1, AaCaMK2 and AaCaMK3 with Setosphaeria turcica CAK1, CAK2 and CAK3 were as high as 94.32%, 97.49% and 86.57%, respectively; RT-qPCR analysis showed that genes expression of AaCaMK1, AaCaMK2 and AaCaMK3 were all significantly upregulated during infection structure differentiation of A. alternata induced by hydrophobic and fruit wax coating surfaces (P<0.05), and fruit wax showed more significant stimulus effects. Among them, the expression of AaCaMK1 and AaCaMK2 during the appressorium formation period (6 h) were 1.51 and 3.05 folds, respectively, while AaCaMK3 had the highest expression in the infection hyphae formation period (8 h), which was 2.86 folds that of the control. Under fruit wax induction, the up-regulated expression of these three genes was significantly higher at the germ tube elongation stage (4 h) on fruit wax coating surfaces than the hydrophobic surface. [Conclusion] AaCaMK in calcium signaling pathway play an important regulatory role on the infection structure differentiation of A. alternata induced by hydrophobic and fruit wax.

Abstract:[Background] There are few reports on the colonization of endophytic control bacteria in faba bean and the diversity of endophytic bacteria. [Objective] To investigate the colonization characteristics of Pseudomonas poae HT1 in faba bean and its effect on the diversity of endophytic bacterial community in the rhizome. [Methods] The colonization characteristics of strain HT1 were determined by antibiotic labeling method, and the community diversity of endophytic bacteria in root and stem of strain HT1 was analyzed by high-throughput sequencing technology. [Results] The number of colonization of strain HT1 was root>stem>leaf, which increased first and then decreased. The root, stem and leaf reached the maximum on the 7th day, and the labeled strain could still be detected on the 83rd day. Root irrigation reduced the richness of endophytic bacteria and increased species diversity of endophytic bacteria in roots. The abundances of Firmicutes, Actinobacteria, Bacteroidetes in roots were significantly increased in irrigated root group, while the relative abundances of Proteobacteria in stems were increased but had no significant effect. At the genus level, after root irrigation, the relative abundance of Romboutsia, Mitsuaria, Lactobacillus, Devosia and other genera increased significantly in the root, and the relative abundance of Pseudomonas, Muribaculaceae, Elstera, Sphingomonas in the stem increased significantly. [Conclusion] The strain HT1 could colonize in faba bean plants for more than 83 days, and could affect the composition of microbial community inside the plants and improve the relative abundance of related microorganisms.

Abstract:[Background] Sclerotinia rot caused by Sclerotinia sclerotiorum is a major disease affecting the yield of sunflower and has occurred frequently in Inner Mongolia and Gansu of China in recent years. [Objective] In order to discover antagonistic strains and effective methods for the biocontrol of sunflower sclerotinia rot. [Methods] The microorganisms in the rhizosphere soil of healthy sunflower plants were isolated by dilution plating method with 4 different media, and then plate confrontation experiments were conducted to select the strains with antagonistic effects on S. sclerotiorum. The selected antagonistic strains were then tested for the control effect with detached leaves. They were identified based on the morphological characteristics, physiological and biochemical properties, and 16S rRNA gene sequence analysis. Finally, we mixed these strains at different ratios into different microbial inoculants for pot experiment to determine the in vivo control effects. [Results] A total of 142 strains of bacteria were isolated from the soils, among which 12 antagonistic strains with an obvious inhibition zone were screened out. Bacillus sp. NM63, JQ134, J7, J33, and Streptomyces sp. Z9 and ZX6 exhibited the inhibition zone diameters greater than 25 mm and significant control effects in detached leaves. Strains NM63, JQ134, J7, Z9, J33, and ZX6 exhibited the control effects of 79.06%, 74.10%, 70.72%, 67.83%, 65.11%, and 57.11% on potted sunflower seedlings, respectively. The microbial inoculants I and II prepared with strains Z9, NM63, JQ134, and J7 mixed at the ratios of 1:1:1:1 and 1:2:2:1 had the control effects of 81.43% and 85.88%, respectively, for potted sunflower. [Conclusion] The antagonistic strains with strong inhibitory effects on S. sclerotiorum were screened out and identified, and the compound microbial inoculant II had good performance in the prevention and treatment of sunflower sclerotinia rot.

Abstract:[Background] The Xinjiang wild apple are suffering serious diseases, and Trichoderma is an important plant disease biocontrol fungus, so it can be used to prevent and control wild apple diseases in Xinjiang. [Objective] In order to isolate and identify the Trichoderma isolates from wild fruit forests in Xinjiang, determine their antifungal ability against two pathogens of wild apples (Malus sieversii), and provide the biocontrol fungal resources for the preparation of “Special Trichoderma Biofertilizer for Xinjiang wild apple”. [Methods] Trichoderma strains were isolated by plate dilution method from the rhizosphere of 4 herbaceous plants in Xinjiang wild fruit forest. Species identification was performed by morphology and molecular biology, and the antifungal ability of Trichoderma isolates was tested by plate confrontation tests, and the solid-state fermentation formula of Trichoderma strains was optimized. [Results] The results show that 2 Trichoderma species (total 42 strains) were isolated, including 34 T. asperellum isolates and 8 T. atroviride isolates. The inhibitory rates of T. asperellum T01 (TasT01) against Colletotrichum gloeosporioides (Cgl) and Alternaria alternaria f. sp. mali (Aal) were 58.60% and 57.14%, respectively; and the inhibitory rates of T. atroviride (TatT35) to Cgl and Aal were 43.86% and 36.90%, respectively. Further microscopic observation showed that TasT01 could inhibit the growth of both pathogens through hypha coiling. The solid-state fermentation formula of TatT35 strain was 90% rice husk, 10% wheat bran, 0.5% (NH₄)₂SO₄. And the spore concentration could reach 1.0×10⁸/g spores. [Conclusion] TasT01 and TatT35 have good inhibitory effects on the two pathogens of Xinjiang wild apple, and the spore concentration significantly increased after the solid-state fermentation formula was optimized. The results provide a reference for biological control of fungal diseases of Xinjiang wild apple.

Abstract:[Background] The nitrogen fertilizer input for sugarcane crop production is very high in China, which result in the high planting costs and cause the field pollution. Sugarcane can obtain high levels of nitrogen via N₂-fixation by associated N₂-fixing bacteria. It is imperative to substantially reduce the nitrogen fertilizer inputs and to substitute nitrogen fertilizers with biological nitrogen fixation to develop a high benefit-cost ratio and environmentally benign sugarcane production in China. [Objective] The aim of this study was to systematically identify efficient endophytic nitrogen fixing bacteria of sugarcane, as well as demonstrate their potential for associative nitrogen fixation and plant growth promotion ability. [Methods] An endophytic diazotroph strain GXS16 was isolated from the roots of sugarcane, and its nitrogen fixation ability was tested by acetylene reduction assay and nifH gene sequence amplification. Strain GXS16 species classification was identified by its culture and the microscopic observation characters, Biolog detection and 16S rRNA gene sequence analysis together. Strain GXS16 was inoculated with micro propagated sugarcane seedlings to detect its growth promotion ability and associative nitrogen fixation activity with ¹⁵N isotope dilution method. [Results] Strain GXS16 showed a high acetylene reduction activity of 2.42 µmol-C₂H₄/(h·mL). Based on phenotypic characteristics, Biolog system test, 16S rRNA gene, nifH and acdS gene sequence analysis, GXS16 was classified into Burkholderia sp. Furthermore, GXS16 containing ACC deaminase activity, and other plant growth promoting traits as IAA production and phosphate solubilization ability. GXS16 significantly promoted the growth of sugarcane seedlings. Compared with the uninoculated controls, GXS16 increased plant height and dry weights more than 15% and 20%, respectively. ¹⁵N isotope dilution assays demonstrated that the associative nitrogen fixation rates of GXS16 in the sugarcane roots, stems and leaves were 7.69%, 15.64% and 8.72%, respectively, which were higher than that of model strain G. diazotrophicus PAL5. [Conclusion] Strain GXS16 has a great potential to efficiently fix N₂ in sugarcane and promote sugarcane growth.

Abstract:[Background] Lactobacillus plantarum is a kind of important probiotic. Our previous study suggested that L. plantarum CCFM8724 could inhibit the double species biofilm of Streptococcus mutans and Candida albicans. However, the specific contributing substances remain unclear. [Objective] To evaluate the characteristics of the antibacterial components and explore the material basis of L. plantarum CCFM8724. [Methods] The influence of temperature, pH and other factors on metabolites were measured. Gas chromatography-mass spectrometry (GC-MS) was performed to analyze the composition of L. plantarum metabolites. Organic solvent extraction and ultrafiltration were further applied to initially separate and purify the fermentation supernatant. The metabolites inhibiting the double species biofilms were then identified by liquid chromatography-mass spectrometry (LC-MS). [Results] Through multivariate statistical analysis, the main difference metabolites of L. plantarum fermentation supernatant were organic acids (such as phenyllactic acid, acetic acid, hydroxyhexanoic acid and glyceric acid, etc.). After preliminary extraction, identification and function verification, the active ingredients were mainly organic acids and cyclic peptide compounds. [Conclusion] The supernatant of L. plantarum CCFM8724 can inhibit the mixed-biofilm through the synergistic effect of organic acids and cyclic peptides. This study provides a theoretical basis for the further production and application of L. plantarum effective components.

Abstract:[Background] Respiratory diseases are common in captive musk deer, some of which are caused by Bordetella bronchiseptica (Bb). However, the research on Bb from forest musk deer (FMD) is insufficient. [Objective] To identify a strain of Bb isolated from the nasal mucus of FMD, analyze its whole-genome sequence and lay a foundation for the prevention and control of related diseases in FMD. [Methods] The pathogen was purified by culture method and preliminarily identified based on colony morphology and biochemical properties. Then, drug sensitivity test and mouse pathogenicity test were carried out to analyze the drug resistance and virulence phenotype. Finally, the whole genome was sequenced. The genetic relationship among species was evaluated based on average nucleotide identity (ANI) at the whole genome level. Meanwhile, gene function annotation and phylogenetic analysis were conducted. [Results] The ANI comparison demonstrated that the pathogen belonged to the classical Bordetella subspecies. According to the colony morphology and biochemical properties, the pathogen was identified as Bb and named FMDBb1. The strain was resistant to lincomycin, rifampicin and most β-lactam antibiotics, with LD₅₀=8.55×10⁶ CFU. The whole genome of the strain was 5 133 936 bp, and gene function annotation showed that it had strong metabolic capacity. In the genome, 65 classical Bordetella virulence factors and the resistance genes targeting pulvomycin and rifampicin were identified. Meanwhile, 19 insertion sequences and 1 phage region were found as well. The strain showed the sequence type 33 and the clone complex 6. The phylogenetic tree constructed based on the housekeeping genes showed that FMDBb1 had the highest homology with KVNON-570 isolated from Korean shorthair cat. [Conclusion] The whole genome of Bb isolated from FMD was sequenced and analyzed, which provided valuable information for the prevention and control of the pathogen infection.

Abstract:[Background] Streptococcus equi subsp. zooepidemicus (SeZ) not only causes swine Streptococcus disease, but also is an important pathogen that causes strangles. Strangle poses a serious threat to the development of horse and donkey industry in China and it is of great significance to strengthen the molecular epidemiological monitoring of the local SeZ isolates for effective prevention and control of strangle. [Objective] To identify the bacteria and detect drug susceptibility, samples were collected from horse farms in Xinjiang. Molecular epidemic and genetic evolution characteristics of the three endemic isolates were also analyzed. [Methods] Isolation and identification, physiological and biochemical test, drug sensitivity test of 3 isolates (ZHZ113, ZHZ211 and ZHZ523), genetic evolution analysis of 16S rRNA and SeM genes were performed. Seven housekeeping genes (arcC, nrdE, proS, spi, tdk, tpi, yqiL) of 3 isolates were amplified and analyzed through multilocus sequence typing (MLST). [Results] Drug sensitivity results showed that all the three isolates were sensitive to 11 drugs like ceftiofuroxime, cefoxitin, gentamicin, streptomycin, erythromycin, doxycycline, levofloxacin, ciprofloxacin, rifampicin, clindamycin and oxytetracycline. The 16S rRNA gene sequence analysis showed that these three isolates belonged to group II (Streptococcus zooepidemicus). The MLST results showed that 3 sequence types ST39, ST419 and ST421 were found and ST419 and ST421 were identified as novel ST genotypes, which have not been reported. From the analysis of SeM gene sequence and the phylogenetic tree we found that there are variations and dynamic changes in the strangles epidemic distribution from the point of view of different countries, different animals and different time. [Conclusion] This study showed that the 3 isolates were closely related to American canine and horse strains and the results provide valuable information to understand the genotype distribution and molecular epidemic characteristics of SeZ in Xinjiang.

Abstract:[Background] Riemerella anatipestifer (RA) is one of the most serious bacterial diseases in water poultry industry. The incidence of Rimerella anatipestifer from geese is increasing, it also brings great economic losses in goose industry. [Objective] To better prevent Rimerella anatipestitis infection in the geese, solve the problems of clinical medication in goose farms and further clarify the relationship between the duck- and goose-origin of RA strains. [Methods] Bacteria were isolated from moribund or dead geese in Jiangsu province and identified by multiple polymerase chain reaction (PCR) and biochemical test. Ten RA strains isolated from the geese were tested for the drug susceptibility, serotypes and pathogenicity in ducks. [Results] Drug sensitivity test showed that majority of the 10 strains from geese were sensitive to mesomycin, sulfafurazole, ceftriaxone, cefradine and florfenicol, but resistance to neomycin, kanamycin, amikacin, gentamicin and clindamycin. All ten strains were multi-drug resistance to 15-25 tested drugs. Serotype 2 was the main serotype of 10 Rimerella anatipestifer isolated from geese in Jiangsu province. Pathogenicity tests on ducklings showed that all the Rimerella anatipestitis isolates from geese caused different degrees of disease in ducklings, and the mortality rate of 3 clinical isolates from geese after 1×10⁷ CFU/feathering challenge was 100%. [Conclusion] This study provides a basis for the prevention, control and clinical treatment of Rimerella anatipestifer from geese in Jiangsu, as well as the further study of the relationship between Rimerella anatipestifer between the duck- and goose-origin of RA strains.

Abstract:[Background] Nocardia seriolae is the causative agent of chronic granulomatous disease in cultured fish characterized by skin ulcers, bleeding and nodular formation in tissues, which seriously harms aquaculture industry. So far, there is no effective prevention and treatment method for the disease. [Objective] To clarify the pathogen causing sarcoidosis of Micropterus salmonoides in an aquaculture farm in Linquan county, Anhui province and to explore its pathogenicity, so as to provide a scientific basis for the effective prevention and treatment of the disease. [Methods] The liver nodules were inoculated with TSB medium to isolate dominant bacterial strain, which was then identified by phenotypic examination combined with test of molecular biology. The pathogenicity of the isolated strain was analyzed through virulence gene identification, LD₅₀ to Micropterus salmonoides as well as the histopathological changes and tissue bacterial load. [Results] A dominant strain NI was isolated from diseased fish and identified as N. seriolae based on its phenotypic characteristics, the 16S rRNA gene sequence identity between the strain NI and N. seriolae reference strains, and the result of specific PCR amplification. Strain NI carried virulence genes gapA, ibeA and mip, and its LD₅₀ to Micropterus salmonoides was 2.58×10⁶ CFU/fish. The clinical signs of Micropterus salmonoides after artificial infection with the strain NI were in accordance with those of natural cases. Histopathologically, chronic granulomatous lesions in head kidney, heart, liver, stomach and spleen of diseased fish, loose intestinal muscle layer and shortened intestinal villi, loose and widened muscle fibers in the muscle tissue were observed. qPCR analysis found the descending order of N. seriolae load in the following infected tissues: head kidney, heart, liver, stomach, spleen, intestine and muscle. [Conclusion] Nocardia seriolae is the pathogen causing the outbreak of sarcoidosis in Micropterus salmonoides in the local aquaculture farm. Results of the study on pathogenicity of the isolated strain provided a theoretical basis for the prevention and control of nocardiosis in Micropterus salmonoides.

Abstract:[Background] Aquatic pathogenic bacteria threaten the health of aquatic organisms and restrict the development of aquaculture. The effective prevention and control of bacterial diseases in fish has become an urgent problem in aquaculture. [Objective] To screen out a bacterial strain with inhibitory effect on aquatic pathogenic bacteria, and to study its antibacterial properties and performance in controlling aquatic bacterial diseases. [Methods] The strain was identified after full consideration of the phylogenetic tree constructed based on the 16S rRNA gene and the physiological and biochemical characteristics. The antibacterial substances were extracted with ethyl acetate. The effects of the strain on the extracellular protease production and biofilm-forming ability of pathogenic bacteria were determined by azo casein method and crystal violet staining, respectively. Furthermore, the inhibitory effect of the strain on Aeromonas veronii Z12 was evaluated by animal experiment. [Results] The lactic acid bacterial strain DH was screened out from the fermented product of pickle and identified as Leuconostoc mesenteroides. The strain secreted extracellular antimicrobial substances, and showed antibacterial effect on Salmonella typhimurium, Escherichia coli, Pseudomonas aeruginosa, Aeromonas salmonicida, Shewanella putrefaciens, and A. veronii. The antibacterial substances could be extracted with ethyl acetate and had thermal stability. Strain DH inhibited the extracellular protease production and biofilm-forming ability of the tested pathogenic bacteria, and exhibited protection against the challenge of Aeromonas veronii Z12. [Conclusion] L. mesenteroides DH can inhibit the growth of aquatic pathogenic bacteria by secreting antibacterial substances, which has the potential for the prevention and control of bacterial diseases in fish.

Abstract:[Background] A fish disease occurred in a snakehead farm in Yangzhou, Jiangsu Province, causes great economic losses. [Objective] To determine the etiology and screen out sensitive drugs for the control of snakehead-related diseases. [Methods] The pathogenic bacteria were isolated from the sick snakeheads, and the isolates were identified in terms of morphology, physiological and biochemical characteristics, 16S rRNA gene and gyrB sequences and specific PCR assays, while their pathogenicity was analyzed by artificial infection tests and antimicrobial sensitivity was tested by paper diffusion method. [Results] A dominant strain SHL isolated from the sick snakeheads was identified as Edwardsiella piscicida, by its morphological characteristics, physicochemical properties, 16S rRNA gene and gyrB sequences, specific PCR results, and further artificial infection tests confirmed its strong pathogenicity to snakeheads with an LD₅₀ of 1.6×10⁵ CFU/g with similar symptoms to natural disease. The drug sensitivity test results showed that the strain was highly sensitive to 28 antibacterial drugs such as penicillin, chloramphenicol and tetracycline, moderately sensitive to erythromycin, and resistant to 6 drugs such as oxacillin, clarithromycin and vancomycin. [Conclusion] Edwardsiella piscicida is the pathogen causing the ulceration and death of snakeheads (Channa argus) cultured in Yangzhou, Jiangsu province. It is the first time that Edwardsiella piscicida was detected from freshwater fish in China. The results demonstrated that the infection spectrum of Edwardsiella piscicida is expanding, which needs to be paid more attention in the field of aquaculture. In aquaculture practice, we can treat the disease with the appropriate national standard fishery drugs according to the results of the drug sensitivity test.

Abstract:[Background] Amanita pallidorosea is a lethal Amanita species in the sect. Phalloideae, containing abundant cyclic peptide toxins. However, its gene family (MSDIN) encoding the toxic peptides and related peptides needs further systematic study. [Objective] This paper aims to explore the diversity, conservation, and phylogeny of genes encoding toxic peptides and related peptides in A. pallidorosea. [Methods] Illumina HiSeq 2000 platform was used for the transcriptome sequencing of A. pallidorosea and TBLASTn for searching MSDIN genes. Specific primers were designed for PCR verification. The types and sequence composition of MSDIN genes were analyzed with the bioinformatics method, and the phylogenetic tree was reconstructed to clarify the evolution. [Results] A total of 60 genes encoding cyclic peptides were obtained from the transcriptome data, which encoded 32 cyclic peptides as verified by PCR, including α-amanitin, β-amanitin, and phallacidin. Among them, 8 new cyclic peptide sequences were reported for the first time in this study. Molecular phylogenetic analysis showed that all cyclic peptides were classified into three clades: amatoxins, phallotoxins, and cyclic peptides of unknown function. Seven potential and new toxic peptides were predicted according to phylogenetic analysis and conserved sequences. [Conclusion] A. pallidorosea boasts abundant cyclic peptides. Transcriptome sequencing can help discover new resources of cyclic peptides in A. pallidorosea, which lays the foundation for the overall structure analysis.

Abstract:[Background] Fusobacterium nucleatum identified in many kinds of tumors is closely related to tumor initiation, poor prognosis, recurrence, and chemotherapy resistance in colorectal tumor. However, the mechanisms of F. nucleatum inducing inflammation and affecting immune cells such as macrophages in tumor microenvironment remain to be elucidated. [Objective] We explored the role and mechanism of F. nucleatum in the process of inducing chronic inflammation and cancer by comparing the monocyte polarization and inflammatory cytokine expression induced by Fusobacterium nucleatum-, Akkermansia muciniphila-, and Escherichia coli-derived lipopolysaccharides (LPSs). [Methods] After the treatment with A. muciniphila LPS, E. coli LPS, F. nucleatum LPS alone or combined with interferon-γ (IFN-γ), we observed the morphological changes of THP-1 and THP-1 (M0) cells. Further, we determined the mRNA levels of macrophage marker genes [including M0 (CD11B), M1 (CD40, CD86), and M2 (CD163, CD206)], TLR3, TLR4, IL-6, and IL-10 as well as the protein levels of IL-6, IL-10, and C-reactive protein. [Results] PAGE results showed that the LPSs from the three microbial species were significantly different in position and number of bands. F. nucleatum LPS possessed stronger activity of inducing adhesion of THP-1 cells. Meanwhile, the group treated with F. nucleatum LPS alone or in combination with IFN-γ had shorter pseudopodia and lower proportion of cells with pseudopodia and spindle-shaped cells (M1 cells) than the groups treated with A. muciniphila LPS and E. coli LPS. The LPSs from A. muciniphila, E. coli, and F. nucleatum up-regulated the mRNA level of CD40 by 5 011.0% (P<0.001), 6 048.9% (P<0.001), and 1 011.6% (P=0.009 4) and that of CD86 by 637.3% (P<0.001), 657.9% (P<0.001), and 194.1% (P>0.05), respectively. The LPSs down-regulated the mRNA level of CD163 by 39.5% (P=0.001 1), 53.7% (P<0.001), and 5.9% (P>0.05) and that of CD206 by 18.6% (P>0.05), 88.4% (P=0.005 5), and 24.8% (P>0.05), respectively. They down-regulated the mRNA level of TLR3 by 32.3% (P=0.044 7), 311.5% (P=0.001 9), and 9.6% (P>0.05), up-regulated that of IL-6 by 17 763.2% (P<0.001), 35 458.2% (P<0.001), and 1 123.6% (P>0.05), and up-regulated that of IL-10 by 729.3% (P<0.001), 1 223.3% (P<0.001), and 124.4% (P>0.05), respectively. The THP-1 cells treated with the LPSs of A. muciniphila, E. coli, and F. nucleatum alone produced IL-6 at 0.16 pg/mL, 6.17 pg/mL, and 0 pg/mL , and those treated with the LPSs in combination with IFN-γ produced IL-6 at 410.03 pg/mL, 1 334.40 pg/mL, and 46.20 pg/mL, respectively. [Conclusion] F. nucleatum LPS possessed a strong activity of recruiting monocytes and inducing them to polarize toward M2. It induced macrophages to produce a much lower amount of IL-6 than the LPSs of A. muciniphila and E. coli, which may play a role in triggering chronic inflammation and tumor immune response and escape. These findings suggest that studying the structure, activity, and mechanism of LPS from carcinogenic, immunomodulating or tumor therapy-associated bacteria will facilitate the elucidation of the role of these bacteria in chronic inflammation and tumorigenesis, which will provide new targets and strategies for the prevention and treatment of these diseases.

Abstract:[Background] S protein is the main structural and immunogenic protein of PEDV. In our previous work, two linear B cell epitopes (BCE) containing short peptides were identified in the cytoplasmic domain of S protein. [Objective] To identify the minimal motif of the linear BCE were identified in the cytoplasmic domain of PEDV S protein. [Methods] Serial 8-mers, advancing one aa once on the short peptide, were prokaryotically expressed. Positive 8-mers were identified by Western Blot using rabbit anti-S serum as the primary antibody and the minimal motif of the linear BCE was identified within the cytoplasmic domain of S protein. [Results] The two short peptides within the cytoplasmic domain of S protein contained the same epitope, and the minimum motif of the epitope was ¹³⁷¹QPYE¹³⁷⁴. Homologous analysis showed that the BCE motif was conserved. [Conclusion] The minimum motif of the BCE within the cytoplasmic domain of S protein was determined as ¹³⁷¹QPYE¹³⁷⁴. Identification of antigen epitope on S protein helps to improve the understanding of its structure and function.

Abstract:[Background] The surface mucilage layer of conidia plays an important role in the interaction between insects and fungi. Hirsutella satumaensis is a host-specific entomopathogenic fungus, and its conidia are covered with a mucilage layer. However, the composition of the mucilage remains unclear. [Objective] The extraction method of mucilage proteins from the conidia of H. satumaensis was optimized, and the extracted proteins were identified by mass spectrometry. [Methods] The extracellular mucilage was eluted with a low concentration of dithiothreitol (DTT) and the extraction conditions were optimized. We used sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to determine the extracted proteins concentrations and propidium iodide (PI) to detect the permeability of conidial membrane. Finally, the proteins from extracellular mucilage were identified by peptide enzymatic hydrolysis, LC-MS/MS, and protein comparative analysis. [Results] The optimized extraction condition was as follows: extraction of the conidia at a concentration of 5×10⁷ cells/mL with 2 mmol/L DTT at 4 ℃ for 24 h, and the cell membrane integrity was not affected after extraction. Seventy-seven proteins were identified from the extracellular mucilage of H. satumaensis conidia, most of which were small molecular secretory proteins, including unannotated proteins, chitinase, lipase, and other insect cuticle-degrading enzymes. [Conclusion] The extracellular mucilage of H. satumaensis has rich proteins involved in host recognition and penetration, which is associated with adhesion and recognition of the host insect.

Abstract:Biological nitrogen removal (BNR) is the key process in wastewater treatment plants, and mastering the metabolic activity of related microorganisms involved in the nitrogen removal process is of great significance for exploring microbial resources potential and improving the nitrogen removal performance of wastewater treatment plants. In recent years, molecular biology methods develop rapidly and has been widely used to reveal the diversity, structure and potential functional genes of the nitrogen removal microorganisms (especially unculturable microorganisms), allowing the role of microorganisms in wastewater BNR system being better clarified. This review introduces the popular molecular biology methods including 16S rRNA gene sequencing, real-time quantitative PCR, metagenomics, metatranscriptomics, metaproteome, and metabolomics, and summarizes their application in nitrifying bacteria, denitrifying bacteria, complete ammonium oxidizing bacteria, anaerobic ammonia oxidizing bacteria, iron-dependent anaerobic ammonium oxidizing bacteria, sulfate-dependent anaerobic ammonium oxidizing bacteria, and nitrite/nitrate type anaerobic methane oxidizing microorganisms, as well as clarifies their metabolic pathways and enzymatic reactions in the nitrogen cycle. It also prospects the technological breakthroughs of molecular biology methods and their application prospects in wastewater BNR systems from three aspects, the “construction of standard determination approaches”, “combination of different methods and interdisciplinary combination” and “simplification of detection methods”. This review provides a comprehensive understanding of the nitrogen removal microbial community and its structure from a systematic perspective, and points out new research directions for future study on BNR microorganisms in wastewater treatment.

Abstract:Enterovirus 71 (EV71) belonging to human enterovirus, is the mainly causative agent of severe Hand, Foot and Mouth Disease (HFMD) and has been associated with various neurological complications, even death. There is currently no effective prevention and specific therapy for EV71, following high disability and mortality rates. Along with the profound study, a large number of compounds with anti-EV71 ability have been discovered, and the mechanisms and drug targets explored are different. Thus, from the perspective of drug to virus and host, this paper reviews and discusses the latest progress in the mechanism of active ingredients in natural drugs, synthetic drugs and common traditional Chinese medicine against EV71 infection. In addition, the screening technology of anti-EV71 drugs was briefly summarized, aiming to provide help for the screening, research and development of anti EV71 drugs.

Abstract:Intestinal microbiota is closely related to colorectal cancer (CRC). The change of intestinal microbial community may be accompanied by the occurrence of CRC, and the emergence of some harmful bacteria may be the direct cause of CRC. Among them, Fusobacterium nucleatum (F. nucleatum), enterotoxigenic Bacteroides fragilis (ETBF) and pks⁺ Escherichia coli (pks⁺ E. coli) were most closely associated with CRC. This review focuses on the pathogenic causes of pks⁺ E. coli and colibactin, the effects on intestinal microbial composition, the synthesis of colibactin and how to inhibit or promote pks⁺ E. coli. At the same time, the possible carcinogenic causes of ETBF and F. nucleatum, their effects on intestinal microbial composition and their promotion or inhibition were also introduced.

Abstract:The abuse of antibiotics and the massive migration of population make pathogenic bacteria resistant to drugs and co-infect with other pathogens, which have posed a serious threat to human life safety. Therefore, the development of new antibacterial drugs has become an urgent problem to be solved. Alanine racemase is a type of isomerases that uses pyridoxal phosphate as the coenzyme to catalyze the optical structure interchange of L-alanine and D-alanine. Its racemic product D-alanine plays a decisive role in the formation of bacterial cell wall, which is closely related to bacterial diseases. Inhibition of the activity of alanine racemase will affect the survival of bacteria. In recent years, it has become an ideal target for the design of antibacterial drugs. The development of inhibitors has become a hot topic in the research and development of antibacterial drugs. This paper systematically elaborated the source, structure, function, application and inhibitor of alanine racemase, and proposed a new strategy for the research of alanine racemase, which would provide a theoretical basis for further research on the relationship between alanine racemase and pathogenic bacteria and the candidate targets of antibacterial drugs.

Abstract:Sucrose phosphorylase (SPase), belonging to the glycoside hydrolase family 13, can catalyze the reversible phosphorolysis of sucrose. Thanks to the broad substrate specificity, SPase can transfer the glucosyl to different acceptors to synthesize arbutin, glycosylglycerol, oligosaccharides, polyphenol derivatives, etc. These products are widely used in food, medicine, cosmetics, and other industries. Owing to the development of enzyme catalysis technology and protein engineering, SPase has received more and more attention, with the application extended. This article reviewed the sources, structures and functions, and application fields of SPase, discussed the protein engineering methods for the enzyme and the limitations, and summarized the future research directions.

Abstract:With the rapid economic growth, a large amount of organic compounds has been released into water bodies, which seriously damages the eco-balance of aquatic system and threatens aquatic life and human health. Remediation via plant-microbe association has attracted wide attention, because of its high efficiency, long duration, low cost, and environmental friendliness. We reviewed the recent advance in the application, research methods, mechanism, and influencing factors of aquatic plant-microbe association in the removal of organic pollutants in water bodies. This review will facilitate the performance optimization of remediation via aquatic plant-microbe association and provide an effective way for the removal of organic pollutants in water bodies.

Abstract:Aspergillus oryzae as an important industrial microorganism, in heterologous protein expression has been widely used. Limited by the modification and secretion process of the expressed protein, the gene donors currently used in actual production are mainly limited to other fungi, especially filamentous fungi. When exogenous genes were derived from plants, insects and mammals, Aspergillus oryzae produced heterologous protein yield and biological activity is often not satisfactory. This paper reviews the research progress of Aspergillus oryzae as a host for expressing heterologous proteins, including its existing genetic manipulation methods and the application and exploration of heterologous expression. In addition, the application prospects and development directions of Aspergillus oryzae expressing heterologous proteins are prospected.

Abstract:The toxin-produing Bacillus cereus is among the common foodborne pathogens. It elicits two types of gastrointestinal illness: vomiting and diarrhoeal syndrome, and causes a variety of local and systemic infections. Due to the extensive use of antibiotics, the resistance of B. cereus has been enhanced, and multiple-antibiotic resistance has been reported. To gain a clear insight into the characteristics and rules of the antibiotic resistance of B. cereus, this paper reviewed the resistance status quo and the mechanism, which is expected to lay a theoretical basis for prevention and control of antibiotic resistance of the bacteria and rational use of antibiotics.

Abstract:In view of the emerging biosafety and bioethics issues in recent years, the teaching of biosafety and bioethics for students majoring in biotechnology is worthy of research and discussion. Small-sized class teaching represents a new measure of teaching reform in China’s colleges and universities in recent years, which is flexible and specific, increases the interactivity between teachers and students, and contributes to the improvement of teaching quality. Considering the characteristics of biosafety and bioethics course in biotechnology specialty and the practice of small-sized class teaching, this paper discusses the teaching experience of small-sized class, analyzes the shortcomings, and puts forward some suggestions for improvement.

Abstract:Environmental Engineering Microbiology is a core course of Environmental Science Specialty. The key points of teaching reform are “Student-Centered” and the cultivation of students’ abilities. Here, the teaching methods are improved by the combination with practice and scientific research, constructing curriculum mind map and integrating ideological education. Practice suggested that the teaching reform in Environmental Engineering Microbiology could increase students’ interest and improve their ability of understanding and applications.

Abstract:A virtual simulation experiment teaching platform for a Microbiology Experimental course was established based on the practical application of Microbiology experiment teaching to study the theme of “isolation and identification of Bacillus subtilis in soil”. This paper describes the construction, function and practical application of the platform. By focusing on the role of modern information technology, this platform can be used with traditional classroom teaching to enable effective improvements in teaching and to enhance students’ interest and ability. This study lays a solid foundation for the promotion of teaching reform and innovation of Microbiology Experiments, and the creation of high-level talents.

Abstract:[Background] Decapod iridescent virus 1 (DIV1), which can infect Penaeus vannamei, Penaeus chinensis, Penaeus japonicus, etc., is one of the main viruses harmful to the prawn aquaculture. Currently, the efficient, rapid, and simple detection of whether the prawn is infected with DIV1 is an effective way to reduce the occurrence and damage of the virus. [Objective] Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas12a), termed CRISPR-Cas12a system, was combined with recombinase polymerase amplification (RPA) to establish a method RPA-Cas12a for the rapid detection of DIV1, and then the application value of this method in detecting actual samples was assessed. [Methods] We extracted the DNA of DIV1, and designed the RPA primers of DIV1, crRNA, and reporting probes to establish the rapid detection method. Subsequently, we analyzed the sensitivity and specificity of this method in detecting DIV1 and compared the consistency of the established method with qPCR. [Results] RPA-Cas12a can detect the DIV1 DNA samples within 40 min and had the sensitivity of 10 copies/reaction. This method only detected DIV1 while showed negative results for white spot syndrome virus, infectious hypodermal and hematopoietic necrosis virus, and Enterocytozoon hepatopenaei. Moreover, the RPA-Cas12a method revealed consistent positive rate with qPCR in detecting 61 actual samples. [Conclusion] The RPA-Cas12a method established in this study is rapid, simple, sensitive, and specific, and can serve as a new tool for the rapid detection of DIV1.

Abstract:[Background] Listeria monocytogenes is a common contaminated foodborne pathogen in meat and dairy products. The traditional culture method can not meet the rapid detection requirements of large quantities of food at the port. It is very important to establish a simple, sensitive, fast and field operable technology. [Objective] To establish a rapid and simple method for the detection of Listeria monocytogenes by recombinase-aided amplification (RAA) to meet the actual needs of port rapid customs clearance and supervision. [Methods] According to the conserved region of hlyA gene of Listeria monocytogenes, the specific primers and probes were designed. The best primer combination with the best amplification efficiency and sensitivity was selected by the combination of primers and probes. The optimal reaction conditions were determined by optimizing the reaction temperature and the concentration of primers and probes. The established fluorescence RAA method was applied to the detection of food matrix and actual samples, and compared with the national standard GB 4789.30-2016. [Results] The optimum reaction temperature of fluorescence RAA of Listeria monocytogenes was 42 ℃, and the final concentration of primer and probe was 400 nmol/L. The established fluorescence RAA method showed high specificity and the sensitivity, and the detection limit of the method was 3×10² CFU/mL in pure culture. The LOD for Listeria monocytogenes was 0.3 CFU/mL, 3 CFU/mL and 30 CFU/mL original concentrations under 4 h LB2 enrichment in artificially contaminated beef, Atlantic salmon and processed cheese respectively. The RAA assay produced a positive signal in 5 min, and the whole assay could be completed in approximately 20-30 min. The speed and sensitivity were significantly higher than those of the national standard method. [Conclusion] The fluorescence RAA method can be used for rapid detection and monitoring of Listeria monocytogenes at ports or other places.

Abstract:[Background] Acute bee paralysis virus (ABPV) is a highly virulent bee virus that causes the death of honeybee and colony exhaustion. [Objective] This experiment was conducted to develop a rapid and sensitive real-time RT-PCR method for the detection of ABPV. [Methods] The primers and probe were designed according to the conservative sequence of capsid protein gene of ABPV registered in GenBank. By optimizing the reaction conditions such as primers, probe concentration, and alternating temperature, a detection method for ABPV based on TaqMan probes was successfully established, and the sensitivity, specificity and stability of the method were tested. [Results] The real-time RT-PCR assay showed a good linear relationship between 9.8×10¹-9.8×10⁸ copies/μL, linear correlation coefficient R² was 0.998, and amplification efficiency was 103.8%. The sensitivity limit of the method was 9.8 copies/μL, and there was no cross-reaction with other honey bee viruses, which represents showing high sensitivity and specificity. The coefficient of variation (CV) for intra-assay and inter-assay repeatability were 0.19%-0.80% and 0.57%-1.07% respectively. In 70 samples of honey bee collected from Fujian regions in 2018 and 2019, the ABPV detection rate was 2.86%. [Conclusion] The ABPV real-time RT-PCR detection method can be used for laboratory testing, epidemiological investigation and epidemic monitoring.