Identification,Isolation and Genomic Characterization of Porcine Astrovirus in Shandong Province,China in 2021-2023

[Objective]The paper was to identify,isolate,and characterize porcine astrovirus in Shandong Province between 2021 and 2023.[Method]A total of 1025 samples of porcine diarrhea samples were collected from various regions of Shandong Province between January 2021 and October 2023.The samples were tested by RT-PCR,followed by sequencing and phylogenetic analyses of the polymerase.[Result]The total positive rate of PAstV was 34.6%(355/1025).The respective proportions of individuals infected with PAstV-1,PAstV-2,PAstV-4 and PAstV-5 were 25.4%(90/355),28.2%(100/355),35.2%(125/355)and 22.5%(80/355),respectively.Additionally,mixed infection was observed.Meanwhile,849 samples of healthy pigs were tested by RT-PCR,and the results demonstrated that the total positive rate of PAstV was 8.13%(69/849).Of these,the proportion of PAstV-1,PAstV-2 and PAstV-4 infection was 27.5%(19/69),37.7%(26/69)and 40.6%(28/69),and a mixed infection also existed.Further sequencing and characterization of some the selected isolates revealed low sequence identities(56.2%)with known PAstV strains,indicating the presence of novel types or genotypes of PAstVs.Furthermore,the isolation conditions of porcine astrovirus were optimized,resulting in the purification of a pure PAstV-4 strain(designated PAstV-4-GRF1).The virus was found to exhibit typical astroviral morphology,with nucleotide identity ranging from 89.9 to 95.4%with previously published PAstV-4 strains.Then,macrovirus transcriptome sequencing showed that 88.30%of the CRF1 samples were mammalian astroviruses.By species classification,PAstV 4 and PAstV 2 accounted for 21.79%and 0.32%,respectively.Phylogenetic tree analysis showed that the c15050 fragment was identical to the GRF-1 sequencing fragment of the isolated strain,and exhibited the highest homology with the Hunan PAstV-4 sequence MK460231 in China.[Conclusion]As the inaugural isolated PAstV-4 strain,it furnishes pivotal materialfor the investigation ofthe biological and pathogenic properties of this virus as well as for the prospectivedevelopment of relevant biological and diagnostic reagents.

Isolation and Identification of Black Spot Pathogen in Psidium guajava

[Objective]The aim was to isolate and identify the pathogen causing black spot disease in guava（Psidium guajava）,so as to determine its taxonomic status.[Method]The fungus was identified by determining its pathogenicity,observing its morphology characteristics and analyzing its ITS sequence.[Result]The pathogen causing black spot disease in guava was a strain of Guignardia mangiferae.[Conclusion]This study will provide theoretical basis for curing black spot disease of guava.

Isolation,Screening and Primary Identification of a Keratin-degrading Fungus

[Objective] The paper was to provide new germplasm sources for efficient and economical degradation and utilization of animal keratin.[Method] The keratin-degrading fungus was isolated,screened and primarily identified by using the combination method of traditional isolation and screening,solid culture-medium degradation and animal test.[Result] A strain of non-pathogenic filamentous fungi with high degradation efficiency was obtained,which was preliminarily identified to be a species in Mucoraceae.[Conclusion] The discovery of the strain enriched the family members of keratin-degrading fungus,and provided new germplasm resources for degradation and utilization of animal keratin.

Isolation,Identification and in vitro Antimicrobial Susceptibility of Pathogenic Aeromonas veronii from Soft-shelled Turtles

In order to effectively control diseases caused by Aeromonas veronii,pathogenic bacteria were isolated and incubated from infected soft-shelled turtles with traditional bacterial isolation method. Four strains of pathogenic bacteria were isolated and identified with traditional biochemical identification method and modern molecular biological identification techniques. According to the results, four strains of pathogenic bacteria were identified as A. veronii biovar sobria. Drug sensitivity test and in vitro antimicrobial test against Bacillus amyloliquefaciens were performed with agar diffusion method. The results showed that B. amyloliquefaciens and several antibiotics such as ofloxacin and gentamicin exerted strong antimicrobial effects on four isolates. B. amyloliquefaciens could be used for the prevention and control of diseases caused by A. veronii in aquaculture.

Isolation and Identification of Anthracnose Pathogen from Fruit of Red Fuji Apple

The fruits of Red Fuji apple with anthracnose symptoms were collected and submitted to tissue isolation and culture. One strain of anthracnose pathogen （numbered as Acgl） was obtained, and it was identified by both morphological and molecular biological methods. According to the morphological characteristics of the colony and conidia and the results of rDNA-ITS sequence analysis, the Acgl strain was identified as Colletotrichum gloeosporioides.

Isolation and Identification of a Flocculant Producing Strain TS-1

[Objective] The purpose was to select out and identify a flocculant producing strain which could produce high active flocculant.[Method] The strain producing high active flocculant was isolated out and purified through medium culture and the selected strain was identified through observing its culture characters and determining its physiological and biochemical property.[Result] Fourteen strains of bacteria with flocculant producing function were isolated from tested soil samples through isolation,purification and preliminary screening using dilution-spread plate method and plate streaking method.Five strains of flocculant producing bacteria showing higher flocculation activity were selected out after second screening and their flocculation rates were higher than 70%;the flocculation activity of one strain among them was still stable after multiple subculturings,its flocculation rate was always above 90% and it was marked as TS-1.TS-1 was encapsulated Gram-positive bacillus and there was no lipid in it,such as poly-β-hydroxybutyric acid.TS-1 was Bacillus amyloliquefaciens,so it was named Bacillus TS-1.[Conclusion] The strain selected out in this experiment could be used in the flocculation and biochemical treatment of wastewater from starch industry.

Isolation of E.tenella JL Strain Single-oocyst and PCR Identification

[Objective] In order to get a purified strain to carry out the relative molecular biology research about E.tenella. [Method] The single-oocyst isolation method was improved and the isolated single-oocyst which was put into capsule was fed to chickens. At the same time, the collected oocysts were identified by PCR method. [Result] The oocysts were isolated from feces of 15 chickens among that of 20 chickens and the infection rate was 75%. The PCR results demonstrated that the single-oocyst strain was E.tenella. [Conclusion] The inoculation of single oocyst capsule was simple, besides, this method did not only save time but also declined inoculation difficulty, increased infection rate and provided good materials for biological research of coccidian.

The Extraction,Isolation and Identification of Exudates from the Roots of Flaveria bidentis

Large amounts ofFlaveria bidentis＇s root culturing solution were obtained by using DFT （deep flow technique） equipment and these solution which was vacuum concentrated （10, 20 mg mL-~） can have a certain inhibition on Triticum aestivum, Cucumis sativus, Raphanus sativus, Amaranthus retroflexus, Setaria viridis, Chenopodium album, Echinochloa crusgalli and Chloris virgata. This outcome suggested some active compounds in the root exudates ofFIaveria bidentis can inhibit the germination, seedling elongation and root length. The dichloromethane extract of root exudates was identificated by GC-MS, and 29 kinds of compounds, including esters, hydrocarbons, ketones, thiazole, amines, etc. were obtained and the phthalate n-octyl ester, phthalate 2-ethylhexyl ester were proved to be allelochemicals. The culturing solution of root exudates was separated through the resin column and silica gel column and a component inhibiting seedling height, root length and fresh weight of wheat was got. There were 6 kinds of organic compounds in this component including dioctyl phthalate, 1,2-phthalate, mono（2-ethylhexyl） ester by GC-MS.

Isolation, Identification and Pathogenicity Analysis of Streptococcus suis Type 2

[Objectives]This study aimed to investigate the pathogenicity,growth characteristics and drug resistance of Streptococcus suis type 2.[Methods]Bacterial isolation and identification,biochemical experiments,determination of growth curve and correlation curve between OD 600 values and viable counts,drug susceptibility tests,pathogenicity analysis,and histopathological observations were carried out.[Results]The Streptococcus strain isolated from infected pigs was identified as Streptococcus suis type 2,which was named TA01 strain.TA01 strain reached the growth peak at 6-8 h post-incubation,and viable counts gradually declined after 8 h of incubation.The correlation equation between OD 600 values and viable counts is y=24.659 x-1.076 1,R^2=0.996 7.TA01 strain was sensitive to penicillin,erythromycin,florfenicol and oxacillin,and resistant to ciprofloxacin,polymyxin B and clindamycin.According to the results of pathogenicity analysis,all the mice in 3.6×10^9 cfu/mouse group died within 48,and these dead mice exhibited acute pyaemia septica.Based on the Reed-Muench formula,it was calculated that LD 50 of TA01 strain was 1.137×10^8 cfu/mouse.Pathological examination showed obvious blue-stained bacteria clusters,accompanied by neutrophil infiltration.[Conclusions]TA01 strain was a virulent strain of Streptococcus suis type 2.Compared with Streptococcus strains which were isolated and reported in China,TA01 strain exhibited strong virulence and rapid proliferation.

Isolation and Identification of High-Quality Lactic Acid Bacteria in Forage Corn

[ Objective] To screen suitable lactic acid bacterium strains from forage corn which can be used as silage additives. [ Method] The lactic acid bacterium strains were isolated by inoculation on MRS solid medium containing calcium carbonate, and they were preliminarily identified through morphological, physiological and biochemical experiments. The acid production efficiency was determined. Twelve strains having strong acid-pro- duction ability were selected, and their salt tolerance and acid tolerance were detected. The sequences of their 16 S rDNA were also analyzed. [ Result] A total of 44 lactic acid bacterium strains were isolated from the forage com. As evidenced by the physiological and biochemical experi- ments, the twelve strains having strong acid-production ability belonged to Leuconostoc, Lactobacillus and Enterococcus, respectively, and they had strong salt tolerance and acid tolerance. According to the sequences of 16 S rDNA, A4, B9, B11, B12 and B14 were Lactobacillus plantarum; A1, A2., A7, A11 and B8 were Leuconostoc mesenteroides dextran subspecies; and AB and A9 were Enterococcus hirae. [ Conclusion ] The lactic acid bacterium strains with strong acid-production ability isolated from forage corn can be developed into silage additives.

Isolation and Identification of Burkholderia glumae from Symptomless Rice Seeds

A survey on isolation and detection of the casual organism of bacterial grain rot of rice was conducted during 1997-2006. In 2006, six pathogenic bacterial strains were isolated from two symptomless seed samples of rice （Oryza sativa L.） originally produced in Hainan Province and then planted in Zhejiang Province, China. They were identified as Burkholderia glumae which is the causal organism of bacterial grain rot of rice by physiological characteristics, colony morphology, pathogenicity test, Biolog, fatty acid methyl ester （FAME） analysis and RAPD-PCR compared with the four standard reference strains. It is confirmed that there is the infection of B. glumae in so-called ＇health looking seeds＇.

Isolation, Identification and Antimicrobial Test in vitro of Aeromonas from Hemibarbus maculatus Bleeker

[Objective] The aim was to quickly find high free drugs to prevent Hemibarbus maculates Bleeker from dying. [Method] The conventional biochemical methods and molecular biological identification method was used to do the isolation and identification of bacteria from H. maculates, and agar diffusion method was used for the susceptibility test and in vitro becteriostasis experiment. The toxins were detected using the plate method, and the intraperitoneal injection method was used to do animal experiments. [Result] Twelve strains of isolated bacteria were obtained, which could uniformly decompose glucose, maltose, but were unable to break down lactose, and they could reduce the nitrate, and were negative in hydrogen sulfide and indole. A clear band was found in 685 bp of the isolated bacteria. And the isolated bacterial all generated hemolysins. Some strains produced protease. The isolated bacterial were resistant to penicillin G, amoxicillin, but sensitive to gentamicin, ciprofloxacin, and could be inhibited by the Bacillus amyloliquefaciens. All experimental animals died within 12 hours. [Conclusion] The 12 isolates were Aeromonas veronii, Aeromonas caviae and Aeromonas hydrophila, respectively, and the deaths of H. maculates were caused by the mixed infection of Aeromonas veronii, Aeromonas caviae and Aeromonas hydrophila. The first choice drug for the treatment was ciprefloxacin, and Bacillus amyloliquefeciens could be used as the drug for ecological prevention and control.

Isolation and identification of alginate-degrading bacteriaand formation of alginase

Five strains of alginate-degrading bacteria Were isolated from the decaying parts of Laminria japonicaand Undaria pinnatifida and identified as Alteromonas espejiana (Strain A101, A102, A103, A105 ) and Alteromonas macleodii(Strain A104). When incubated at 25 t for 144 h in the liquid mediuxn which contamed 0. 5%peptone, 0. 3% -0. 6%sodium alginate, 0. 1 % yeast extract, 3% NaCI, pH 7. 5, the stain A102 pnduced the highest amount of alginase.

Isolation and Structural Identification of Herbicidal Active Substance from Root of Flaveria bident(L.) Kuntze

In order to understand the composition and structure of herbicidal active substance from the root of Flaveria bidentis （L.） Kuntze, the isolation and structural identification were researched in this paper. The crude extract from the root ofF. bidentis （L.） Kuntze was extracted by petroleum ether, ethyl acetate, and water saturation of n-butyl alcohol, respectively, and the extraction fluid was separated by using the method of TLC, then the main fraction was separated by HPLC, and the structure of the herbicidal active substance was analyzed by LC-MS, elemental analysis and ~H-NMR. The results showed that the petroleum extraction had the strongest herbicidal activity, and the purple blue stripe separated by TLC had the strongest effect on Digitaria sanguinalis. The herbicidal active substance was identified as ct-terthienyl according to the data of LC-MS, elemental analysis and 1H-NMR.

Isolation,Identification and Pathogenic Characteristics of Duck Hepatitis Virus Isolates

[ Objective] The aim of this study was to determine and analyze the pathogen and the reasons of duck viral hepatitis which is prevalent recently and difficult to control. [Method] Viruses were isolated from livers and spleens of ducks with typical clinical symptoms in Linyi, Weifang, Binzhou and other regions of Shandong Province. The pathogenic characteristics were observed by inoculation in chicken or duck embryo, RT- PCR, serological test, and duck regression. [ Result] Four duck hepatitis virus （DHV） strains were isolated, and the 5th passage allantoic fluid contained 10^3.41 -10^5.20 ELD50/mI. The serum cross protection rate was 20% -80% between the DHV stains and DHV type I. The mortalities of 4- day-old healthy ducks challenged by these four stains were 50% -100%. All challenged ducks had typical lesions of duck viral hepatitis, and the death peak appeared after 24-48 h. [Conclusion] The virulence of different DHV isolates has regional difference.

Isolation and Identification of Antioxidative Active Component from Total Flavonoids of Mimenghua

In this study, bioassay-guided isolation, identification and biological evaluation of antioxidant components from total flavonoids of Chinese herbal plant Mimenghua were performed. 1, 1-Diphenyl-2-picrylhydrazyl(DPPH) radical scavenging test was adopted as the bioassay-guided method, Sephadex LH-20 column chromatography and high performance liquid chromatography were used as the purification tools for the acquisition of antioxidant components. One compound was obtained and identified as acteoside by its physicochemical properties and spectral characteristics. Antioxidant activity in vitro of acteoside was determined by evaluation of the scavenging activity on DPPH radical and hydroxyl radical, reducing power and total antioxidant capability.The results showed that acteoside had significant antioxidative activity and possessed higher activity with the increase of concentration, which provides the potential application of acteoside in food, pharmaceutical and cosmetic industries.

Isolation and Identification of Photobacterium damselae subsp.damselae(PDD) from Tongue Sole

In May 2016,an epizootic occured among cultured tongue soles caused mass deaths in a fish farm in Qinhuangdao,China.In order to find out the etiological agent,a bacterial strain was isolated from ascites and other tissues of sick tongue sole aseptically collected.The isolate was identified as Photobacterium damselae subsp.damsela（PDD） by isolation culture,Gram staining,physiological identification,morohological observation,biochemical identification and 16S rDNA sequence analysis.The results showed that the isolate shared 99.6% homology with the reference strain in GenBank.The animal regression test displayed that the isolate had very strong pathogenicity to tongue sole.The LD（50） was 3.1 × 10~4 CFU/mL,and it showed pathogenicity to mammals.The antimicrobial susceptibility test showed the isolate was highly sensitive to nrofloxacin,Norfloxacin,Ciprofloxacin,Mequindox;moderately sensitive to Cefradine,Doxycycline;and insensitive to Gentamicin,Ceftriaxone,Tilmicosin,etc..

Isolation and Identification of a Specific cDNA Mapping to the Bam HI-I2 and -LFragments within the Inverted Repeats ofUnique Long Re-gion (IRL) in the Genom e ofMarek′s Disease Herpesvirus (MDV) Oncogenic Strain Beijing-1

Objective\ To understand the transcription of BamHI L DNA fragment from genome of strong virulent GA strain of Marek′s disease herpesvirus (MDV) in lymphoblastoid tumor tissue induced by oncogenic strain Beijing 1 (a specific local strain in China) of MDV. Methods\ Two oligonucleotide primers were synthesized according to the reported sequence of \%meq\% gene an ideal oncogenic candidate and our previously determined sequence of BamHI L fragment of Marek′s disease herpesvirus (MDV), respectively. Reverse transcriptase PCR(RT PCR) assay was performed by using these primers and the mRNA as a template which was isolated from visceral lymphoblastoid tumors obtained from chickens artificially infected with strain Beijing 1 of oncogenic MDV. Southern blot molecular hybridization was further carried out to detect the product of RT PCR with digoxigenin labeled nucleotide probe from BamHI I2 and L fragment in the gene library of MDV strain GA, respectively. Results\ Two probes could simultaneously hybridize this cDNA amplified by RT PCR with a length of about 730 bp. Conclusion\ It is suggested that \%meq\% transcription could extend from the right hand end of BamHI I2 to the adjacent BamHI L, and the BamHI L region was likely to be transcribed in MDV induced lymphoblastoid tumors.

Pathogen Isolation and Identification of a Serious Leaf Disease of Red Sandalwood (Pterocarpus santalinus) Seedlings

In the introduction and propagation of red sandalwood （Pterocarpus santalinus）, a serious leaf disease of its seedlings in winter and spring seasons was found, but the name of the disease and its pathogen species have not been reported. The pathogen isolated from infected leaves of 18-month-old seedlings was identi- fied as Colletotrichum gloeosporioides by morphological characteristics of colony and conidium, and analysis results of rDNA-intemal transcribed spacer sequence （ITS） of the strain. Pathogenicity test results further confirmed that C. gloeosporioides was the pathogen responsible for the infected leaves symptoms of red sandal- wood. However, the disease belongs to an atypical anthraenose. Control of the leaf diseases of red sandalwood seedlings was discussed.

Isolation,Identification and Antibiotics Susceptibility Test of Citrobacter freundii from Procambarus clarkia

This experiment was conducted to clarify species and drug resistance of pathogen from the diseased Procambarus clarkia. Pathogenic bacteria from hepatopancreas of the diseased P. clarkia were examined using conventional methods,and then were isolated. The further tests and analysis of the isolated strain were developed,including the regression experiment to P. clarkia,the morphology,physiological and biochemical characteristics,sequence analysis of their 16 S rRNA and gyr B genes,and the susceptibility test to antibiotics. Large colonies with similar morphology and color were obtained. Strain X120523 was identified as Citrobacter freundii,proved to have strong pathogenicity,and was susceptible to quinolones and aminoglycosides.