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 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.

Fermented Corn Flour Poisoning in Rural Areas of China Ⅲ. Isolation and Identification of Main Toxin Produced by Causal Microorganisms

Flavotoxin A was isolated from Pseudomonas cocovenenans subsp. farinofermentans culture in semisolid potato-dextrose-agar medium, which was isolated from fermented corn meal that had caused food poisoning outbreaks in China. The isolation, purification, and chemical structure of this toxin were studied. The NMR spectra, the uv spectra, and molar extinction coefficients, and the mass spectra of Flavotoxin A are in good agreement with those reported for bongkrekic acid. Therefore, Flavotoxin A and bongkrekic acid are the same organic chemical compound; the molecular formula is C_(28)H_(38)O_7. The oral LD_(50) of the purified Flavotoxin A in mice was 3.16mg/kg (1.53-6.15mg/kg). The existence of bongkrekic acid in toxic fermented corn samples collected during food poisoning outbreaks was also confirmed. It is concluded that bongkrekic acid has played an important role in the outbreaks of fermented corn poisoning. 1989 Academic Press, Inc.

ISOLATION AND IDENTIFICATION OF β-N-OXALO-L-α,β-DIAMINOPROPIONIC ACID AND ITS ISOMER α-N-OXALO-L-α,β-DIAMINOPROPIONIC ACID FROM PANAX SPECIES

The neuroexcitotoxic nonprotein amino acid β-N-oxalo-L-α, β-diaminopropionic acid (β-N-ODAP)and its isomer a-N-oxalo-L-α, β-diaminopropionic acid (α-N-ODAP)in Panax ginseng C. A. Meyer(cultivated ih Northeastern China), Panax quinquefolius L., Panax notoginseng F.H. Chen (cultivated in Southwestern China), Korean red ginseng and Jilin red ginseng were isolated and identified. Efficient separation and purification methods for β-N-ODAP and α-N-ODAP were developed.

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 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 THE RELATED GENE MEDIATING γ-RADIATION-INDUCED APOPTOSIS IN HUMAN HL- 60 CELLS

To identify the member of the caspase family proteases involved in γ radiation induced apoptosis in HL 60 cells, using degenerated oligonucleotide primers encoding the highly conserved peptides, which were present in all known caspases, RT PCR was performed on poly (A) RNA from the γ radiation induced apoptotic HL 60 cells. Then, cloned and sequenced to identify the amplified DNA fragments. The results showed that the amplified DNA fragments were identified with a part of caspase 3 cDNA. It indicated that caspase 3 was involved in γ radiation induced apoptosis in HL 60 cells and may be the pivotal element of radiation induced apoptosis.

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 and Identification of Porcine Epidemic Diarrhea Virus(PEDV) HLJ Strain with IPEC-J2 Cells and Phylogenetic Analysis of Its S Gene

Porcine epidemic diarrhea(PED)is caused by porcine epidemic diarrhea virus(PEDV),and is characterized by vomiting,diarrhea and dehydration of suckling pigs from 80% to 100% morbidity and 50% to 90% mortality,and resulted in tremendous economic losses to swine industry.The PEDV mainly infects small intestine of pigs,resulting in vacuolar degeneration and necrosis of mucosal epithelium.The IPEC-J2 is a pig intestine epithelial cell line,which is similar to the intestinal environment of piglets,can be used to isolate and identify the PEDV field isolates.In this study,it appeared the PEDV typical postmortem changes and histopathological lesion of degeneration and destruction of small intestine in infected piglets,and IHC identified that the PEDV distributed in the mucosa and submucosa of small intestine mostly.Furthermore,the PEDV HLJ strain was successfully isolated and characterized in the IPEC-J2 cells,and indicated that the IPEC-J2 cell line was sensitive to isolate and adapt the PEDV field strain,and could be utilized to multiply the PEDV rapidly.The S gene analysis indicated that the PEDV HLJ strain was the prevailed virus,belonged to Group 1 with attenuated virulent DR13,SC1402 and J-S2/2015 strains isolated in South Korea and China from 2014 to 2015.This study had important theoretical and practical significances on analyzing genetic variation of the PEDV,understanding the pathogenic characteristics of the virus and developing new vaccines for the PED.

Isolation and Identification of three Lactobacilli and Research on Antimicrobial Activity in Vitro

Collected from the broiler cecal contents, bacteria were isolated and purified. These isolated and purified Lactobacilli were detected by acidity and bile salt tolerant test, then three better tolerant strains were screened out. These three strains were conducted to observe the morphous through microscope, perform Gram stain, and to identify their physiological and biochemical characteristics. The results showed that L1 was L.casei, L2 was L.gasseri and L3 was L.graminis. With Oxford Cup method, these three Lactobacilli were detected for their bacteriostasis. Results showed that L1, L2 and L3 strains had strong antibacterial activities to E. coli and Salmonella.

Isolation and Identification of a High-pathogenicity Strepto- coccus suis Serotype 7 Strain

In order to diagnose the diseased pigs in a certain large pig farm in Binzhou City, Shandong Province, the dead piglets with joint swelling were subjected to necroscopy, and the pathogenic bacterium was isolated and identified. One Gram-positive Streptococcus was isolated. The strain was subjected to characteristic culture, microscopic examination and molecular biological identification, and resistance detection, animal regression experiment and mouse pathogenicity test were carried out. The results showed that the isolate was identified to be Streptococcus suis serotype 7, which was resistant to multiple drugs; and the pathogenicity test showed that the strain had high pathogenicity to pigs, resulting in neurosis on partial pigs, and the strain had no pathogenicity to Kunming and BALB/c mice but certain pathogenicity to CD1 mice.

Isolation and Identification of Main Fungal Pathogens in Muskmelon in Open Field of Hubei Province in Summer

Fungal diseases often occur seriously in muskmelon in open field of Hubei Province in summer, especially in continuous cropping pattern, resulting in great economic losses. In this study, the pathogens of main fungal diseases in muskmelon in open field of Hubei Province were isolated, and they were identified by morphological and molecular techniques. The results showed that muskmelon fusarium wilt is a major disease in muskmelon in open field of Hubei Province in summer, and its pathogen was confirmed to be Fusarium oxysporum. In future studies, one pair of specific primers would be designed to detect different pathogenic races of Fusarium oxysporum so as to accelerate the detection and to shorten the detection time,thereby proving guidance for actual production.

Isolation and Identification of Different Sources of Salmonella and Comparisons on the Drug Resistance Spectrum Types

[ Objective] In order to make out differences between drug resistance spectrum types of different animal sources of Salmonella. [ Meth- otis] Selenite cystine broth, Salmonella- Shigella genus agar medium, CHROMagar Salmonella chromogenic culture, Reveal Salmonella detection kit and molecular biology methods were used for isolation and identification of Salmonella from different animal feces samples. Antibacterialantimi- crobial susceptibility tests of 17 kinds of drugs such as enrofloxacin, apramycin and florfenicol were done on isolated strains with broth microdilution method, and test data was analysed with WHOnet 5.4 software. [ Results ] 699 feces samples from different animals were separated, and 53 strains of Salmonella were achieved with separation rate of 7.58%, among which those of yak, poultry, and pet sources were respectively 4.33%, 9.06% and 8.37%. [ Condusions] Separation rates and drug resistance spectrum types of different animal origin Salmonella varied greatly, among which separation rates and drug resistance spectrum types of yak source Salmonella were the lowest, there was little difference between separation rates of poultry and dog source Salmonella, however there were large differences on drug resistance spectrum types.

Isolation and Identification of Haemophilus parasuis SD02 strain

A bacterial strain was isolated from the sick pigs suspiciously infected by polyserositis and arthritis in a pig farm in Shandong Province, and identified through morphological observation, culture traits, bioehemical characteristics and PCR amplifieation. Additionally, primers were de- signed according to the 16S rRNA sequence of Haemophihzs parasuis, and the bacterial strain was amplified by PCR. The amplified fragments of approximately 1 400 bp was sequenced, and aligned with the sequence in GenBank. The results showed that it shared the homology of 97%-99% with the 16S rRNA sequence of foreign H.parasuis, and confirmed as H.parasuis （HPS）. The strain was determined as serotype 4 through serotype identification. The strain was named SD02.

Isolation and Identification of Pathogenic Bacteria Causing Bovine Subclinical Mastitis in Eastern Hebei Province

In this study, the milk samples of 1 021 cows in eight dairy farms in Eastern Hebei Province were collected and detected with LMT reagent and somatic cell count for subclinical mastitis. Pathogenic bacteria in subclinical mastitis positive milk samples were isolated and identified.The results showed that 60.63%（619/1 021） of the sampled cows were diagnosed with subclinical mastitis, and mixed infections accounted for 88.21%（546/619） of the cases. In addition, 82 strains of 14 species were isolated from the subclinical mastitis positive milk samples, including 36 strains of Staphylococcus（43.90%）, 33 strains of Streptococcus（40.24%）, 8 strains of Enterobacteriaceae（9.76%） and 5 strains of Corynebacterium（6.10%）, respectively. The results proved that Staphylococcus aureus and Streptococcus agalactiae are the main pathogenic bacteria causing bovine subclinical mastitis in Eastern Hebei Province.

Isolation and Identification of Staphylococcus chromogenes and 3 D Structural Analysis of the katA Gene

[Objective]Staphylococcus arthritis became an increasingly significant health problem in intensive chicken farming in China.[Method]In this study,a bacteria strain was isolated from the broiler chicken suffering from arthritis and named as the strain Gg1.[Result]It was then identified as Staphylococcus chromogenes by the biochemical tests and phylogenetic tree analysis based on 16S rDNA sequence.Furthermore,the catalase（katA）gene was amplified by PCR using the designed primers,and the expected fragment was 1 232 bp long encoding a protein of 410 amino acids that shares the conserved motifs including catalase,heme-binding ligand and active center motif.Six phosphorylation sites（Ser95,Thr96,Ser241,Ser242,Thr281,Ser338）,four conserved residues（Ser95,His216,Tyr281,Asp341）and two active sites（His56,Asn129）were demonstrated by multiple sequence alignment and homology comparisons.The homology modeling of 3D structure of katA protein was done by SWISSMODEL server based on the template retrieved from the catalase（PDB：2ISA_A）of Vibrio salmonicida.The katA protein represents a four-domain globular protein,the quality and reliability of the resulting protein structure was further verified by Ramachandran plot.[Conclusion]To our knowledge,this is the first report of S.chromogenes linked to arthritis in chicken and the bioinformatic characterization of its katA gene.

Isolation and Identification of Pathogenic Fungi Causing Decayin Luotian Chestnut during Late Storage Period and Research on Pathogenicity

In order to explore moldy mechanism of chestnut from Luotian County in storage process,the strains of pathogenic fungi were isolated from chestnuts after storage at room temperature for 70d.Six genera of fungi were found in chestnut through experimental identification,which were Ozoniumsp.,Fusarium sp.,Aspergillus sp.,Penicilliumsp.,Rhiopus sp.and Stachybotrys sp.,respectively.The re-inoculation tests had been conducted on pathogenic fungi whose isolating rate was greater than 10%.The result showed that the rest genera of fungi generally had no pathogenicity except Penicilliumsp.could infect non-injured chestnut with a lower moldy rate and lighter symptoms;but the moldy rate of strains was above 60% in injured inoculation and they showed heavy symptoms,among which the moldy rate of Ozoniumsp.and Aspergillus sp.were higher than 80%.The experimental results showed that injured chestnut were more likely to decay.Ozoniumsp.and Aspergillus sp.were important pathogenic fungi causing decay during storage process of chestnut.

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.

Identification and Antibacterial Activity Study of a Terrestrial Strain of Brevibacterium aureus 431

This study isolated and purified strain 431 from an animal probiotic product.Through staining and microscopic examination,colony morphology analysis,biochemical reaction tests,and 16S rDNA sequence alignment,the strain was identified and named Brevibacterium aureus 431.The study focused on the production of biosurfactants by strain 431,and antibacterial activity tests were conducted on the strain and its secondary metabolites.The results showed that strain 431 exhibited no resistance to 10 commonly used drugs,and its concentrated secondary metabolites were highly sensitive to the indicator bacterium Escherichia coli.Oral administration of strain 431 to BALB/c mice resulted in normal mental state,diet,and bowel movements,with no signs of illness or death,indicating that strain 431 is highly safe and non-pathogenic to mice.The study suggests that Brevibacterium aureus 431 has significant research value as a new source of actinomycetes and that its secondary metabolites have potential application value in the development of antibacterial drugs.

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.