Development of a High-throughput Sequencing Platform for Detection of Viral Encephalitis Pathogens Based on Amplicon Sequencing

Objective Viral encephalitis is an infectious disease severely affecting human health.It is caused by a wide variety of viral pathogens,including herpes viruses,flaviviruses,enteroviruses,and other viruses.The laboratory diagnosis of viral encephalitis is a worldwide challenge.Recently,high-throughput sequencing technology has provided new tools for diagnosing central nervous system infections.Thus,In this study,we established a multipathogen detection platform for viral encephalitis based on amplicon sequencing.Methods We designed nine pairs of specific polymerase chain reaction(PCR)primers for the 12 viruses by reviewing the relevant literature.The detection ability of the primers was verified by software simulation and the detection of known positive samples.Amplicon sequencing was used to validate the samples,and consistency was compared with Sanger sequencing.Results The results showed that the target sequences of various pathogens were obtained at a coverage depth level greater than 20×,and the sequence lengths were consistent with the sizes of the predicted amplicons.The sequences were verified using the National Center for Biotechnology Information BLAST,and all results were consistent with the results of Sanger sequencing.Conclusion Amplicon-based high-throughput sequencing technology is feasible as a supplementary method for the pathogenic detection of viral encephalitis.It is also a useful tool for the high-volume screening of clinical samples.

Catalog of operational taxonomic units and unified amplicon sequencing data for the microbiomes of medicinal plant roots

China has a rich history of cultivating medicinal plants,whose root microbial communities closely interact with the medicinal plants,thereby influencing their growth,health,and medicinal properties.Currently,researchers widely use 16S rRNA gene amplicon sequencing to study these root microbial communities.However,publicly available sequence datasets often lack essential sample information or contain errors,impeding the reuse of the datasets in the future.In this study,we aimed to create a united,reliable,and readily usable source of 16S rRNA gene sequences for medicinal plant root microbiomes.We compiled a catalog of 1392 microbiome samples for 58 medicinal plants from 58 studies,and manually provided essential sample information based on the experimental setup described in the associated papers.We then processed the sequences using a custom pipeline,generating a united catalog of operational taxonomic units(OTUs)and conducting taxonomic classification.We also pre-dicted the ecological functions of the communities for each sample.Finally,we used this dataset,to compare the rhizosphere bacterial communities of Pseudostellaria heterophylla from Fujian and Guizhou Provinces,revealing significant differences in the community composition of the same plant from different geographic locations.By providing a comprehensive and united catalog of amplicon sequences and OTUs for medicinal plant root bacterial communities,this study offers an invaluable resource for future comparative studies and data mining.

Diversity and dynamics of microbial communities in brown planthopper at different developmental stages revealed by high-throughput amplicon sequencing

The microbiome associated with brown planthopper(BPH)plays an important role in mediating host health and fitness.Characterization of the microbial community and its structure is prerequisite for understanding the intricate symbiotic relationships between microbes and host insect.Here,we investigated the bacterial and fungal communities of BPH at different developmental stages using high-throughput amplicon sequencing.Our results revealed that both the bacterial and fungal communities were diverse and dynamic during BPH development.The bacterial communities were generally richer than fungi in each developmental stage.At 97%similarly,19 phyla and 278 genera of bacteria were an-notated,while five fungal phyla comprising 80 genera were assigned.The highest species richness for the bacterial communities was detected in the nymphal stage.The taxonomic diversity of the fungal communities in female adults was generally at a relatively higher level when compared to other developmental stages.The most dominant phylum of bacteria and fungi at each developmental stage all belonged to Proteobacteria and Ascomycota,re-spectively.A significantly lower abundance of bacterial genus Acinetobacter was recorded in the egg stage when compared to other developmental stages,while the dominant fun-gal genus Wallemia was more abundant in the nymph and adult stages than in the egg stage.Additionally,the microbial composition differed between male and female adults,suggesting that the microbial communties In BPH were gender-dependent.Uverall,our study enriches our knowledge on the microbial communities associated with BPH and will provide clues to develop potential biocontrol techniques against this rice pest.

Spatiotemporal dynamics of the microbial diversity on salt-preserved goatskins assessed by culturing and 16S rRNA gene amplicon sequencing

Wet-salted skin,as a special artificial high-salt environment,is rich in protein,fat,collagen and other nutrient substrates,and is a rich resource of halotolerant and halophilic microorganisms.However,knowledge gaps regarding the microbial community structure and inter taxa associations of wet-salted skin are large.In this study,the spatiotemporal dynamics and community structure of microorganisms present on wet-salted goatskins were investigated using 16S rRNA gene amplicon sequencing and culturable technique.Alpha diversity analysis based on Sobs,Chao,Ace and Shannon indices revealed that microbial diversity on the wet-salted goatskins exhibited a trend of‘down→up→down→flat’with time.During preservation,genera belonging to the bacteria domain such as Aci-netobacter,Weissella and Streptococcus were slowly dying out,whereas those belonging to halophilic archaea such as Natrialba and Haloterrigena were gradually flourishing.Moreover,to resist high-salt stress,microorganisms on the wet-salted goatskin gradually migrated from the outside to the inside,eventually leading to the microbial diversity inside the skin being the same as or even higher than that on the skin surface.Venn diagram analysis revealed that the strains of some genera,including Psychrobacter,Salimicrobium,Salinicola,Ornithinibacillus,Halomonas,Bacillus and Chromohalobacter,were distributed throughout the interior and exterior of the wet-salted goatskin and existed during various periods.Accordingly,45 protease-producing halophilic or halotolerant microorganisms were isolated and screened from the wet-salted goatskin using the gradient dilution plate method.Importantly,16S rRNA genes of some bacteria exhibited less than 99.5%similarity to valid published species,indicating that they likely are novel spe-cies and have a good potential for application.

Periodic Addition of Glucose Suppressed Cyanobacterial Abundance in Additive Lake Water Samples during the Entire Bloom Season

Previously, we showed that prophylactic addition of glucose to Harsha Lake water samples could inhibit cyanobacteria growth, at least for a short period of time. The current study tested cyanobacterial control with glucose for the entire Harsha Lake bloom season. Water samples (1000 ml) were collected weekly from Harsha Lake during the algal-bloom season starting June 9 and lasting until August 24, 2022. To each of two 7-liter polypropylene containers, 500 ml of Harsha Lake water was added, and the containers were placed in a controlled environment chamber. To one container labeled “Treated,” 0.15 g of glucose was added, and nothing was added to the container labeled “Control.” After that, three 25 ml samples from each container were collected and used for 16S rRNA gene sequencing each week. Then 1000 ml of Harsha Lake water was newly collected each week, with 500 ml added to each container, along with the addition of 0.15 g glucose to the “Treated” container. Sequencing data were used to examine differences in the composition of bacterial communities between Treated and Control containers. Treatment with glucose altered the microbial communities by 1) reducing taxonomic diversity, 2) largely eliminating cyanobacterial taxa, and 3) increasing the relative abundance of subsets of non-cyanobacterial taxa (such as Proteobacteria and Actinobacteriota). These effects were observed across time despite weekly inputs derived directly from Lake water. The addition of glucose to a container receiving weekly additions of Lake water suppressed the cyanobacterial populations during the entire summer bloom season. The glucose appears to stimulate the diversity of certain bacterial taxa at the expense of the cyanobacteria.

Alterations in gut microbiota are related to metabolite profiles in spinal cord injury

Studies have shown that gut microbiota metabolites can enter the central nervous system via the blood-spinal cord barrier and cause neuroinflammation, thus constituting secondary injury after spinal cord injury. To investigate the correlation between gut microbiota and metabolites and the possible mechanism underlying the effects of gut microbiota on secondary injury after spinal cord injury, in this study, we established mouse models of T8–T10 traumatic spinal cord injury. We used 16 S rRNA gene amplicon sequencing and metabolomics to reveal the changes in gut microbiota and metabolites in fecal samples from the mouse model. Results showed a severe gut microbiota disturbance after spinal cord injury, which included marked increases in pro-inflammatory bacteria, such as Shigella, Bacteroides, Rikenella, Staphylococcus, and Mucispirillum and decreases in anti-inflammatory bacteria, such as Lactobacillus, Allobaculum, and Sutterella. Meanwhile, we identified 27 metabolites that decreased and 320 metabolites that increased in the injured spinal cord. Combined with pathway enrichment analysis, five markedly differential amino acids(L-leucine, L-methionine, L-phenylalanine, L-isoleucine and L-valine) were screened out, which play a pivotal role in activating oxidative stress and inflammatory responses following spinal cord injury. Integrated correlation analysis indicated that the alteration of gut microbiota was related to the differences in amino acids, which suggests that disturbances in gut microbiota might participate in the secondary injury through the accumulation of partial metabolites that activate oxidative stress and inflammatory responses. Findings from this study provide a new theoretical basis for improving the secondary injury after spinal cord injury through fecal microbial transplantation.

Diversity and geographical distribution of haptophyte Phaeocystis in the Chinese seas based on metabarcoding analysis

Phaeocystis is an important bloom-forming species and over 100 blooms have occurred since 1997 along the Chinese coasts,while only the species P.globosa was described.In this project,a total of 246 phytoplankton samples collected from the Bohai Sea,the Yellow Sea,and the East China Sea in April 2021 were analyzed to evaluate the species diversity and geographical distribution of the genus Phaeocystis based on metabarcoding 18S r DNA sequence.Four described Phaeocystis species were recognized,including P.globosa,P.pouchetii,P.jahnii,and P.cordata,and each has distinguished geographical distribution characteristics.P.globosa was the most widespread and abundant species,and P.pouchetii was found in the Bohai Sea and the northern Yellow Sea with substantially elevated abundance.P.cordata was also a widespread species,but its abundance was relatively low,while P.jahnii gathered only in the southeastern East China Sea.Water temperature,phosphate as well as ammonium contents were found to be associated with the abundance of P.globosa,P.pouchetii,and P.jahnii significantly.Moreover,two uncharacterized Phaeocystis species were detected in the Chinese seas,indicating the diversity of the genus remains underestimated worldwide.

Supplementation of a lacto-fermented rapeseed-seaweed blend promotes gut microbial-and gut immune-modulation in weaner piglets

Background:The direct use of medical zinc oxide in feed will be abandoned after 2022 in Europe,leaving an urgent need for substitutes to prevent post-weaning disorders.Results:This study investigated the effect of using rapeseed-seaweed blend(rapeseed meal added two brown macroalgae species Ascophylum nodosum and Saccharina latissima)fermented by lactobacilli(FRS)as feed ingredients in piglet weaning.From d 28 of life to d 85,the piglets were fed one of three different feeding regimens(n=230 each)with inclusion of 0%,2.5% and 5% FRS.In this period,no significant difference of piglet performance was found among the three groups.From a subset of piglets(n=10 from each treatment),blood samples for hematology,biochemistry and immunoglobulin analysis,colon digesta for microbiome analysis,and jejunum and colon tissues for histopathological analyses were collected.The piglets fed with 2.5% FRS manifested alleviated intraepithelial and stromal lymphocytes infiltration in the gut,enhanced colon mucosa barrier relative to the 0% FRS group.The colon microbiota composition was determined using V3 and V1-V8 region 16S rRNA gene amplicon sequencing by Illumina NextSeq and Oxford Nanopore MinION,respectively.The two amplicon sequencing strategies showed high consistency between the detected bacteria.Both sequencing strategies indicated that inclusion of FRS reshaped the colon microbiome of weaned piglets with increased Shannon diversity.Prevotella stercorea was verified by both methods to be more abundant in the piglets supplied with FRS feed,and its abundance was positively correlated with colonic mucosa thickness but negatively correlated with blood concentrations of leucocytes and IgG.Conclusions:FRS supplementation relieved the gut lymphocyte infiltration of the weaned piglets,improved the colon mucosa barrier with altered microbiota composition.Increasing the dietary inclusion of FRS from 2.5% to 5% did not lead to further improvements.

Influence of group B streptococcus and vaginal cleanliness on the vaginal microbiome of pregnant women

BACKGROUND The vaginal microbiome plays a critical role in the health of pregnant women and their newborns.Group B Streptococcus(GBS)and vaginal cleanliness significantly affect the vaginal microecosystem and are closely associated with vaginal diseases.AIM To explore the effects of GBS status and vaginal cleanliness on vaginal microecosystems.METHODS We collected 160 vaginal swabs from pregnant women and divided them into the following four groups based on GBS status and vaginal cleanliness:GBS-positive+vaginal cleanliness I–II degree,GBS-negative+vaginal cleanliness I–II degree,GBS-positive+vaginal cleanliness III–IV degree,and GBS-negative+vaginal cleanliness III–IV degree.Samples were subjected to 16S rRNA gene amplicon sequencing.RESULTS Alpha diversity analysis showed that the Shannon index did not significantly differ between the four groups.We identified significant variation in taxa abundance between the GBS-positive and GBS-negative groups and between the vaginal cleanliness I–II degree and III–IV degree groups.Principal coordinate analysis and non-metric multidimensional scaling analysis further confirmed the microbial diversity of the four groups.Moreover,the linear discriminant analysis demonstrated that Lactobacillus jensenii and Actinobacteria were strongly associated with GBS-positive status,and Lactobacillus iners,Lactobacillaceae,Lactobacillus,Lactobacillales,Bacilli and Firmicutes were closely correlated with GBS-negative status.CONCLUSION GBS status and vaginal cleanliness significantly affect vaginal microbiome differences in pregnant women.Our findings provide instructional information for clinical antibiotic treatment in pregnant women with different GBS statuses and vaginal cleanliness degrees.

Fecal microbiome of growing pigs fed a cereal based diet including chicory(Cichorium intybus L.) or ribwort(Plantago lanceolata L.) forage

Background： The purpose of this study was to investigate how inclusion of chicory forage or ribwort forage in a cereal-based diet influenced the fecal microbial community（microbiome） in newly weaned（35 days of age） piglets.The piglets were fed a cereal-based diet without（B） and with inclusion（80 and 160 g/kg air-dry forage） of vegetative shoots of chicory（C） and leaves of ribwort（R） forage in a 35-day growth trial. Fecal samples were collected at the start（D0）, 17（D17） and 35（D35） days after weaning and profiles of the microbial consortia were generated using terminal restriction fragment length polymorphism（T-RFLP）. 454-FLX pyrosequencing of 16 S r RNA gene amplicons was used to analyze the microbial composition in a subset of the samples already analyzed with T-RFLP.Results： The microbial clustering pattern was primarily dependent on age of the pigs, but diet effects could also be observed. Lactobacilli and enterobacteria were more abundant at D0, whereas the genera Streptococcus, Treponema,Clostridium, Clostridiaceae1 and Coprococcus were present in higher abundances at D35. Pigs fed ribwort had an increased abundance of sequences classified as Treponema and a reduction in lactobacilli. However, the abundance of Prevotellaceae increased with age in on both the chicory and the ribwort diet. Moreover, there were significant correlations between the abundance of Bacteroides and the digested amount of galactose, uronic acids and total non-starch polysaccharides, and between the abundance of Bacteroidales and the digested amount of xylose.Conclusion： This study demonstrated that both chicory and ribwort inclusion in the diet of newly weaned pigs influenced the composition of the fecal microbiota and that digestion of specific dietary components was correlated with species composition of the microbiota. Moreover, this study showed that the gut will be exposed to a dramatic shift in the microbial community structure several weeks after weaning.

Effects of enrichmemt planting with native tree species on bacterial community structure and potential impact on Eucalyptus plantations in southern China

Multi-generational planting of Eucalyptus species degrades soil quality but the introduction of legumes can improve soil fertility and microbial diversity.However,the effects of introducing non-legume native tree species on soil nutrients and bacterial community structure remain poorly understood.This study investigated the impacts of the conversion of third generation monoculture Eucalyptus plantations to mixed systems including Eucalyptus urograndis with Cinnamomum camphora(EC)and E.urograndis with Castanopsis hystrix(EH),on soil chemical and biochemical properties and bacterial community structure,diversity and functions.First generation E.urophylla plantations were the control.Results show that planting the third generation Eucalyptus led to a significant decrease in p H,organic matter,nutrient content,enzyme activities(invertin,acid phosphataes,and urease),and bacterialα-diversity compare to the controls.However,the mixed planting showed significant improvement in soil chemical and biochemical attributes and bacterialα-diversity,although the E.urograndis and C.hystrix planting had no improvement.Chloroflexi(oligotrophic bacteria)were significantly enriched in third generation Eucalyptus and Eucalyptus+C.hystrix,while proteobacteria increased significantly in the E.urograndis with C.camphora plantings.The relative abundance of multiple metabolic pathways increased significantly in the third generation Eucalyptus plantations whereas membrane transportrelated genes were enriched in soils of the mixed systems.The changes in bacterial community structures in the two mixed systems were driven by diversity,organic matter and acid phosphatase,while bacterial functions were affected by invertase,NO_(3)^(-)-N,diversity and urease.These results suggest that the transformation of successive monoculture Eucalyptus plantations into mixed plantations reduces the depletion of soil nutrients and enhances the ecological function of soil microorganisms.

Predicted Functional Shifts Due to Type of Soil Microbiome and Watering of Two Wild Plants in Western Region of Saudi Arabia

The present study aimed to predict differential enrichment of pathways and compounds in the rhizosphere microbiomes of the two wild plants(Abutilon fruticosum and Nitrosalsola vermiculata)and to predict functional shifts in microbiomes due to water.Amplicon sequencing of 16S rRNA region V3–V4 was done and gene-based microbial compositions were enrolled in PICRUSt to predict enriched pathways and compounds.The results indicated that“ABC transporters”and“Quorum sensing”pathways are among the highest enriched pathways in rhizosphere microbiomes of the two wild plants compared with those of the bulk soil microbiomes.The highest enriched compounds in soil microbiomes of the two wild plants included five proteins and three enzymes participating in one or more KEGG pathways.Six of these eight compounds showed higher predicted enrichment in rhizosphere soil microbiomes,while only one,namely phosphate transport system substrate-binding protein,showed higher enrichment in the surrounding bulk soil microbiomes.In terms of differentially enriched compounds due to watering,only the dual-specific aspartyl-tRNA(Asn)/glutamyl-tRNA(Gln)amidotransferase subunit A showed higher enrichment in rhizosphere soil of the two wild plants after 24 h of watering.Two of the highly enriched compounds namely branched-chain amino acid transport system ATP-binding protein and branched-chain amino acid transport system substrate-binding protein,are encoded by genes stimulated by the plant’s GABA that participates in conferring biotic and abiotic stresses in plants and improves the plant’s growth performance.The 3-Oxoacyl-[ACP]reductase,a member of the short-chain alcohol dehydrogenase/reductase(SDR)superfamily,participates in fatty acids elongation cycles and contributes to plant-microbe symbiotic relationships,while enoyl-CoA hydratase has a reverse action as it participates in“Fatty acid degradation”pathway.The methyl-accepting chemotaxis protein is an environmental signal that sense“Bacterial chemotaxis”pathway to help establishing symbiosis with plant roots by recruiting/colonizing of microbial partners(symbionts)to plant rhizosphere.This information justifies the high enrichment of compounds in plant rhizosphere.The dual-specific aspartyl-tRNA(Asn)/glutamyl-tRNA(Gln)amidotransferase subunit A contributes to the plant ability to respond to watering as it participates in attaching the correct amino acid during translation to its cognate tRNA species,while hydrolyzing incorrectly attached amino acid.These two actions reduce the influence of oxidative stress in generating misfolded proteins and in reducing fidelity of translation.

Characterization of Fe(Ⅲ)-Reducing Enrichment Cultures and Isolation of Enterobacter sp. Nan-1 from the Deep-Sea Sediment, South China Sea

To characterize the Fe(III)-reducing bacteria,enrichment cultures were initiated by inoculating deep-sea sediment from the South China Sea(SCS)into the media with hydrous ferric oxide(HFO)as the sole electron acceptor.As indicated by Meta 16S rDNA Amplicon Sequencing,the microorganisms related to Fe(III)-reduction in the enrichment cultures were mainly Shewanella and Enterobacter.A new facultative Fe(III)-reducing bacterium was obtained and identified as Enterobacter sp.Nan-1 based on its 16S rRNA gene sequence and physiological characterizations.Enterobacter sp.Nan-1 was not only a mesophilic bacterium capable of reducing HFO with a wide range of salinity(4,34,40,50 and 60 g L−1)efficiently,but also a piezotolerant bacterium that can proceed Fe(III)-reduction sustainedly at hydrostatic pressures between 0.1 and 50 MPa using glucose and pyruvate as carbon source.Furthermore,the geochemical characteristics of deep-sea sediment indicated that the microbial metabolism and iron reduction both remain active in the well-developed Fe(III)-reducing zone where the strain Nan-1 was obtained.To our knowledge,Enterobacter sp.Nan-1 could serve as a new applicative Fe(III)-reducing bacterium for future investigation on the iron biogeochemical cycle and diagenetic process of organic matter in the deep-sea environment.

Application of Biological Nanopore Sequencing Technology in the Detection of Microorganisms

Environmental pollution and the spread of pathogenic microorganisms pose a significant threat to the health of humans and the planet.Thus,understanding and detecting microorganisms is crucial for maintaining a healthy living environment.Nanopore sequencing is a single-molecule detection method developed in the 1990s that has revolutionized various research fields.It offers several advantages over traditional sequencing methods,including low cost,label-free,time-saving detection speed,long sequencing reading,real-time monitoring,convenient carrying,and other significant advantages.In this review,we summarize the technical principles and characteristics of nanopore sequencing and discuss its applications in amplicon sequencing,metagenome sequencing,and whole-genome sequencing of environmental microorganisms,as well as its in situ application under some special circumstances.We also analyze the advantages and challenges of nanopore sequencing in microbiology research.Overall,nanopore sequencing has the potential to greatly enhance the detection and understanding of microorganisms in environmental research,but further developments are needed to overcome the current challenges.

Microbiome Diversity Analysis of the Bacterial Community in Idah River, Kogi State, Nigeria

The analysis of bacterial diversity in aquatic systems particularly in rivers, lakes, and streams can provide useful data on the effect of anthropogenic activities on such water bodies to humans and fishes. Idah River, the focal point of this study, is an offshoot of the two major Nigerian rivers characterized by observed human activities and pollution sources. Water samples were collected from four designated sites and assessed for their bacterial assemblages and structure, using PacBio Single-Molecule Real-Time (SMRT) sequencing technology. The full length of the 16S rRNA gene was sequenced, and Amplicon Sequence Variants were generated using the DADA2 workflow optimised for PacBio long-read amplicons in Rstudio. A total of 8751 high-quality reads obtained were taxonomically classified as 24 phyla, 42 classes, 84 orders, 125 families, 156 genera, and 106 species. Taxonomical composition revealed Proteobacteria as the most abundant phyla across all sample sites. At the genera level, Azospira (57.03%) was the most dominant ASV in Docking Point A, while Acinetobacter (66.67%) was the most abundant ASV in Docking Point B. In Idah Axis Confluence, hgcl clade (65.66%) was the most prevalent ASV, whereas Holophaga (42.86%) was the most common ASV in Idah Axis Midstream. Genera analysis also revealed that 12.9% of the total ASVs were discovered across all sample sites. Among these were pathogenic bacteria, reducers, and degraders of domestic and animal wastes. Observed results provide evidence that sampled sites of Idah River are contaminated, most likely through constant human activities and thus, could have an impact on resident fishes as well. This study, therefore, agrees with a previous report from the river, which used standard microbial procedures. However, next-generation sequencing techniques employed revealed more bacterial community than the former, including unresolved taxonomic sequences that may be novel.

Mutations of voltage-gated sodium channel contribute to pyrethroid resistance in Panonychus citri

Insecticide resistance in Panonychus citri is a major obstacle to mite control in citrus orchards.Pyrethroid insecticides are continually used to control mites in China,although resistance to pyrethroids has evolved in some populations.Here,the resistance to the pyrethroid fenpropathrin was investigated and 7 out of 8 field-collected populations of P citri exhibited a high level of resistance,ranging from 171-fold to 15391-fold higher than the susceptible(SS)comparison strain.Three voltage-gated sodium channel(VGSC)mutations were identified in the tested populations:L1031V,F1747L,and F17511.Amplicon sequencing was used to evaluate the frequency of these mutations in the 19 field populations.L1031V and F1751I were present in all populations at frequencies of 11.6%-82.1%and 0.5%-31.8%,respectively,whereas the F1747L mutation was only present in 12 populations from Chongqing,Sichuan,Guangxi,and Yunnan provinces.Introduction of these mutations singly or in combination into transgenic flies significantly increased their resistance to fenpropathrin and these flies also exhibited reduced mortality after exposure to the pyrethroids permethrin andβ-cypermethrin.Panonychus citri VGSC homology modeling and ligand docking indicate that F1747 and F1751 form direct binding contacts with pyrethroids,which are lost with mutation,whereas L1031 mutation may diminish pyrethroid effects through an allosteric mechanism.Overall,the results provide molecular markers for monitoring pest resistance to pyrethroids and offer new insights into the basis of pyrethroid actions on sodium channels.

Isolation of exogenous fungi from Notoginseng Radix et Rhizoma and preliminary analysis of toxigenic fungi

Notoginseng Radix et Rhizoma(Sanqi in Chinese)is a precious traditional Chinese herbal medicine.It has the effect of dispersing blood stasis and stopping bleeding,reducing swelling and fixing pain.However,it tends to contaminate with harmful fungi during storage,which may make it much less effective.In order to understand the fungal contamination of Notoginseng Radix et Rhizoma and master its composition of the exogenous fungi.The surface fungi of Notoginseng Radix et Rhizoma samples collected from six Chinese provinces and districts were investigated by using dilution plate method.Detection of aflatoxins by UPLC-MS/MS.The results showed that Penicillium citrinum was dominantly isolated from Notoginseng Radix et Rhizoma samples from No.1 to No.4.Aspergillus flavus,which produces aflatoxin,was dominantly isolated from Notoginseng Radix et Rhizoma samples from No.5 and No.6.In addition,kinds of mycotoxin were assayed which were produced by three of those identified A.flavus.All three fungi strains produced aflatoxin B1(AFB1)and one strain HBSQ1-5 additionally produced other three kinds of mycotoxin,AFB2,AFG1 and AFG2.It is the results implied that it will be very important to take serious cautions when using Notoginseng Radix et Rhizoma.As well as,understanding the composition of the exogenous fungi of Notoginseng Radix et Rhizoma and the strains of toxin-producing fungi,which can play an important role in guiding the storage of Notoginseng Radix et Rhizoma.

Effects of nanofertilizer and nano-plant hormone on soil chemical properties and microbial community in two different soil types

Application of nanotechnology in agriculture has been expanded to improve crop production.The impact of nanomaterials(NMs)on factors that influence the survival and function of beneficial microorganisms is a less studied aspect that needs to be better understood.Only a few studies have assessed the effects of NMs on beneficial soil microorganisms.This study was conducted to assess the effects of nanofertilizer FertiGroe?N(FG-N)and nano-plant hormone HormoGroe?auxin(HG-A)on the chemical properties and microbial communities of two contrasting soils,Lipa clay loam(CL)and Sariaya sandy loam(SL),over a 35-d incubation period in the laboratory.Bacterial and fungal communities were evaluated using amplicon sequencing analysis within the 16S and internal transcribed spacer regions,respectively.The application of FG-N significantly decreased soil pH,but did not affect total N and available P for both soil types.A significant increase in exchangeable K was observed only in Lipa CL.The application of HG-A had no significant effect on soil chemical properties.Regarding the bacterial community after incubation,the relative abundances of Acidobacteriia,Chthonomonadetes,and Saccharimonadia decreased,whereas Acidimicrobiia,Chloroflexia,and Gemmatimonadetes increased with FG-N application in Lipa CL.The application of HG-A increased the relative abundance of Rubrobacteria,Chthonomonadetes,and Chloroflexia in Lipa CL.For the fungal community,FG-N application increased the relative abundance of Sordariomycetes,Agaricomycetes,and Eurotiomycetes,whereas Dothideomycetes and Mortierellomycetes decreased in Lipa CL after incubation.In Sariaya SL,FG-N application increased the relative abundance of Dothideomycetes,Eurotiomycetes,and Mortierellomycetes,but decreased that of Sordariomycetes and Agaricomycetes.Fungal classes observed in the control samples were not detected in the HG-A treatment,but were recovered after incubation in Lipa CL.The microbial diversity in both soil types showed slight changes with FG-N and HG-A application.Principal coordinate analysis illustrated the clustering of bacterial and fungal taxa between Lipa CL and Sariaya SL.Pearson correlation analysis showed that several bacterial and fungal communities were positively or negatively correlated with soil pH.The results suggest that FG-N can be safely used in crop production and HG-A may be used mainly for vegetative propagation.

Coastal mudflats as reservoirs of extracellular antibiotic resistance genes: Studies in Eastern China

Despite coastal mudflats serving as essential ecological zones interconnecting terrestrial/freshwater and marine systems,little is known about the profiles of antibiotic resistance genes(ARGs)in this area.In this study,characteristics of typical ARGs,involving both intracellular(iARGs)and extracellular ARGs(eARGs)at different physical states,were explored in over 1000 km of coastal mudflats in Eastern China.Results indicated the presence of iARGs and eARGs at states of both freely present or attached by particles.The abundance of eARGs was significantly higher than that of iARGs(87.3%vs 12.7%),and their dominance was more significant than those in other habitats(52.7%-76.3%).ARG abundance,especially for eARGs,showed an increasing trend(p<0.05)from southern(Nantong)to northern(Lianyungang)coastalmudflats.Higher salinity facilitated the transformation from iARGs to eARGs,and smaller soil particle size was conducive to the persistence of eARGs in northern coastal mudflats.This study addresses the neglected function of coastal mudflats as eARGs reservoirs.

Changes in soil free-living diazotrophic community and co-occurrence patterns along desert wetland degradation gradient in the Mu Us Desert,northern China

Climate change and human activity have led to the degradation of desert wetlands.Free-living diazotrophs are vital for soil nitrogen input.However,a comprehensive understanding of how soil free-living diazotrophic communities and their co-occurrence patterns respond to desert wetland degradation is lacking.Here,quantitative polymerase chain reaction(qPCR),amplicon sequencing targeting nitrogenase gene(nifH),and network analysis were used to investigate the abundance,diversity,community composition,and co-occurrence patterns of soil free-living diazotrophs along the wetland degradation gradient,i.e.,non-degraded(ND),lightly degraded(LD),moderately degraded(MD),and severely degraded(SD),in the southeastern Mu Us Desert,northern China.The abundance and Shannon,Simpson,Chao 1,and ACE indexes decreased(P<0.05)by 14.6%,20.7%,2.1%,46.5%,and 45.0%,respectively,in SD wetland,whereas no significant difference(P>0.05)was observed between ND and LD wetlands.The relative abundance of Proteobacteria generally decreased(by 53.5%–19.7%)across the different degradation levels,while the relative abundance of Cyanobacteria increased(by 6.2%–40.1%)from ND to MD levels.The abundance,diversity,and community composition of diazotrophs were most strongly related to soil organic carbon,followed by total nitrogen,moisture,and pH.The least number of network nodes and edges and the lowest density were observed for MD and SD wetlands,indicating that the complexity of free-living diazotrophic networks was reduced by continued degeneration.Overall,severe desert wetland degradation affected the abundance,diversity,and network complexity of soil free-living diazotrophs more negatively than light degradation.This degradation promoted the growth of autotrophic diazotrophs and inhibited the growth of heterotrophic diazotrophs.These changes were mostly related to the loss of soil organic carbon.