• The Plant Pathology Journal
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• v.27 no.1
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• pp.44-52
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• 2011

Field surveys were conducted in 45 stone fruit orchards in seven districts of Isparta Province located in western Mediterranean region of Turkey important for stone fruit production. Leaf samples were collected from 175 trees showing virus-like symptoms. These samples were first tested by ELISA for five different RNA viruses including Apple mosaic ilarvirus (ApMV), Prunus necrotic ringspot ilarvirus (PNRSV), Prune dwarf ilarvirus (PDV), Plum pox potyvirus (PPV), Apple chlorotic leafspot trichovirus (ACLSV). While no ApMV and PPV infection was found, 46, 24 and 16 samples were tested positive for PDV, ACLSV and PNRSV, respectively, in ELISA showing about 45% of symptomatic trees in the region were infected with at least one of these viruses. In addition, it was found that nine sweet cherry trees were mixed infected with two or three of these viruses and PDV with an infection rate of 26.3% was the most widespread virus in symptomatic trees in western Mediterranean region. Thirty samples were selected and tested by a multiplex RT-PCR (mRT-PCR) for simultaneous detection of these viruses. While PPV was not detected, more than half of the tested 20 samples were individually or mixed infected with ApMV, ACLSV, PNRSV and PDV. The mRT-PCR results were confirmed by detection of these viruses individually in some of the field samples using RT-PCR with primes specific to each virus. Comparison of ELSA and mRT-PCR results of 30 samples showed that numbers of infected and mixed infected samples as well as infection and mixed infection rates were significantly higher in RT-PCR (20 and 66.7%) than in ELISA (14 and 46.7%). The results confirm that mRT-PCR is more sensitive than ELISA.

• The Plant Pathology Journal
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• v.19 no.3
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• pp.159-161
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• 2003

The coat protein (CP) gene of Apple mosaic virus (ApMV), a member of the genus Ilarvirus, was selected for the design of virus-specific primers for amplification and molecular detection of the virus in cultivated apple. A combined assay of reverse transcription and polymerase chain reaction (RT-PCR) was performed with a single pair of ApMV-specific primers and crude nucleic acid extracts from virus-infected apple for rapid detection of the virus. The PCR product was verified by restriction mapping analysis and by sequence determination. The lowest concentration of template viral RNA required for detection was 100 fg. This indicates that the RT-PCR for detection of the virus is a 10$^3$times more sensitive, reproducible and time-saving method than the enzyme-linked immunosorbent assay. The specificity of the primers was verified using other unrelated viral RNAs. No PCR product was observed when Cucumber mosaic virus (Cucumovirus) or a crude extract of healthy apple was used as a template in RT-PCR with the same primers. The PCR product (669 bp) of the CP gene of the virus was cloned into the plasmid vector and result-ant recombinant (pAPCP1) was selected for molecule of apple transformation to breed virus-resistant transgenic apple plants as the next step. This method can be useful for early stage screening of in vitro plantlet and genetic resources of resistant cultivar of apple plants.

• Proceedings of the Zoological Society Korea Conference
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• 1995.10b
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• pp.341.1-341
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• 1995

No Abstract, See Full Text

• KOREAN JOURNAL OF CROP SCIENCE
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• v.65 no.4
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• pp.496-507
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• 2020

Virus infections of apples result in lowered commercial qualities such as low sugar content, weakened tree vigor, and malformed fruits. An effective way to control viruses is to produce virus-free plants based on the development of an accurate and sensitive diagnostic method. In this study, real-time PCR assays based on SYBR Green I and TaqMan probes were developed for detecting ASGV, ASPV, and ApMV viruses. These methods can detect and quantify 10³ to 10¹¹ RNA copies/μL of each virus separately. Compared with methods with two different dyes, the SYBR Green I-based method was efficient for virus detection as well as for assay using the TaqMan probe. Field tests demonstrated that real-time PCR methods developed in this study were applicable to high-throughput diagnoses for virus research and plant quarantine.

• The Plant Pathology Journal
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• v.18 no.5
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• pp.259-265
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• 2002

Apple mosaic virus (ApMV), a member of the genus Ilarvirus, was detected and isolated from diseased 'Fuji' apple (Malus domestica) in Korea. The coat protein (CP) genes of two ApMV strains, denoted as ApMV-Kl and ApMV-K2, were amplified by using the reverse transcription and polymerase chain reaction (RT-PCR) and were analyzed thereafter. The objectives were to define the molecular variability of genomic information of ApMV found in Korea and to develop virus-derived resistant gene source for making virus-resistant trans-genic apple. RT-PCR amplicons for the APMVS were cloned and their nucleotide sequences were determined. The CPs of ApMV-Kl and ApMV-K2 consisted of 222 and 232 amino acid residues, respectively. The identities of the CPs of the two Korean APMVS were 93.1% and 85.6% at the nucleotide and amino acid sequences, respectively. The CP of ApMV-Kl showed 46.1-100% and 43.2-100% identities to eight different ApMV strains at the nucleotide and amino acid levels, respectively. When ApMV-PV32 strain was not included in the analysis, ApMV strains shared over 83.0% and 78.6% homologies at the nucleotide and amino acid levels, respectively. ApMV strains showed heterogeneity in CP size and sequence variability. Most of the amino acid residue differences were located at the N-termini of the strains of ApMV, whereas, the middle regions and C-termini were remarkably conserved. The APMVS were 17.(1-54.5% identical with three other species of the genus Ilarviyus. ApMV strains can be classified into three subgroups (subgroups I, II, and III) based on the phylogenetic analysis of CP gene in both nucleotide and amino acid levels. Interestingly, all the strains of subgroup I were isolated from apple plants, while the strains of subgroups II and III were originated from peach, hop, or pear, The results suggest that ApMV strains co-evolved with their host plants, which may have resulted in the CP heterogeneity.

• Journal of Plant Biotechnology
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• v.6 no.2
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• pp.69-75
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• 2004

Optimal protocol for efficient genetic transformation has been defined to aid future strategies of genetic engineering in pigeon pea with agronomically important genes. Transgenic pigeonpea plants were successfully produced through Agrobacterium tumefaciens-mediated genetic transformation method using cotyledonary node explants by employing defined culture media. The explants were co-cultivated with A. tumefaciens strain C-58 harboring the binary plasmid, pCAMBIA-1301 [con-ferring $\beta$-glucuronidase(GUS) activity and resistance to hygromycin] and cultured on selection medium (regeneration medium supplemented with hygromycin) to select putatively transformed shoots. The shoots were then rooted on root induction medium and transferred to pots containing sand and soil mixture in the ratio of 1:1. About 22 putative TO transgenic plants have been produced. Stable expression and integration of the transgenes in the putative transgenics were confirmed by GUS assay, PCR and Southern blot hybridization with a transformation efficiency of over 45%. Stable integration and expression of the marker gene has been confirmed in the TO and T1 transgenics through PCR, and Southern hybridization.

• Korean Journal of Veterinary Research
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• v.42 no.3
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• pp.363-370
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• 2002

For the development of diagnostic polymerase chain reaction (PCR) to fungal infection by dermatophytes Trichophyton and Microsporum, detection of the fungal DNA by PCR and analysis of the DNA pattern were undertaken in the present study. A total of 15 strains were tested and those consisted of 3 reference strains and 12 isolates such as: reference strains of T mentagrophytes (downy type, ATCC 9533), T rubrum (IFO 6204) and M gypseum (ATCC 9083), and each isolate of T mentogrophytes (powdery type), T mentagrophytes (granular type), T mentogrophytes (purple-red type), T rubrum, T raubitschekii, T tonsurans, T equinum, T ajelloi, T verrucosum, M cookei, M nanum and M gypseum. The DNA were purely isolated from all strains of Trichophyton spp. and Microsporum spp. by a simple method partly consisted of disruption of fungal cells by lyophilization and grinding and extraction of fungal DNA without phenol treatment which is a routine procedure in DNA isolation. For the detection of fungal DNAs, optimal condition of PCR was determined as preheating once at $94^{\circ}C$ for 5 min, 35 cycles of denaturation at $94^{\circ}C$ for 1 min, annealing at $38^{\circ}C$ for 1 min and polymerization at $72^{\circ}C$ for 2 min, and 1 cycle of final extension at $72^{\circ}C$ for 5 min. In PCR using arbitrary primers AP-1 (5' ACCCGACCTG3') and AP-2 (5' ACGGGCCAGT3'), DNAs in various numbers and sizes were detected from different species of Trichophyton and Microsporum, while DNAs in similar size were also detected in all strains of Trichophyton spp. and Microsporum spp. There were unique DNAs observed from certain dermatophytes by AP-1 such as 1,900 bases in T rubrum, 950 and 1,100 bases in T raubitscheldi, 2,100 bases in T equinum, 400 bases in T verrucosum and 1,150 bases in M gypseum. The unique DNAs were also observed by AP-2 such as 1,200 bases in T ajelloi, 250 bases in T verrucosum, 1,150 bases in M cookei and 2,000 bases in M nanum. The results indicated that PCR can detect a specific DNA from certain Trychophyton and Microsporum spp, which can be the information for further development of diagoomc PCR to dennatophytes.

• The Korean Journal of Mycology
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• v.26 no.4 s.87
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• pp.466-477
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• 1998

Twenty-two Phellinus strains were characterized using colony morphologies and polymerase chain reaction (PCR) to divide into Phellinus linteus. There were some differences in mycelial growth and colony shapes among the strains when they were grown on various media such as PDA, MCM, MEA and YM. Phellinus linteus was slowly growing, formed golden-yellow colony, and produced blue pigment on PDA media. When the regions of internal transcribed spacer (ITS) were amplified from ribosomal RNA (rRNA) coding genes of P. igniarius and P. linteus strains by means of PCR, two types of band (700 bp and 800 bp) were appeared, respectively. For the amplified intergenic region I (IGRI), P. igniarius strains showed a different band among 500, 600, 700 and 800 bp according to the strains, whereas P. linteus strains did one specific band of 700 bp. By polymorphism analysis after digesting the amplified products with 6 different restriction enzymes, a band specific to P. linteus was generated when the products for ITS region were digested with HaeIII, suggesting that the enzyme digestion could provide effective method to distinguish between P. igniarius and P. linteus. And also, the analysis of genetic relationship showed that the genetic similarities were 89% and 95% in P. igniarius and P. linteus strains, respectively. Random amplification polymorphic DNA (RAPD) analysis using multiple primer sets and arbitrarily primed PCR (AP-PCR) with ITS3 primer could also result in a reproducible way to identify P. linteus strains.

• The Korea Journal of Herbology
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• v.28 no.3
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• pp.95-106
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• 2013

Objectives : The researcher investigated the anti-obesity effect of Alismatis Orientale Rhizoma(AP) water extract in mice fed a high fat diet and focused on the analysis of local area adipose tissue. Methods : Male ICR mice were divided into three groups, which were fed either a normal AIN diet, a 45% high fat diet (CT group), or a high fat diet and orally administration with a concentrations of 100 mg/kg (AP100 group) and 300 mg/kg body weight (AP300 group) for eight weeks. Results : As compared with CT group, AP100 group showed significant reductions in absolute weight of liver. As compared with CT group, AP100 group and AP300 group showed significant reductions in weight gain and relative weight of total fat. AST, triglyceride, total-cholesterol levels from the AP 100 group and 300 group were significantly lower than those of the CT, and ALT, LDL-cholesterol levels from the AP 100 group was significantly lower than those of the CT. But serum HDL-cholesterol levels from the AP 100 group and 300 group were significantly higher than those of the CT. And serum adiponectin levels from the AP 100 group was significantly higher than those of the CT. In result of real time PCR, all mRNA expression(PEPCK, G6Pase, PGC-$1{\alpha}$ and $ERR{\gamma}$) of two experimental groups were significantly decreased compared to those of CT group. The treatment with AP on local abdominal area made a fat cell size lessen on the fat tissue in the abdominal cavity and subcutaneous area. Conclusions : These results suggest that AP has an anti-obesity effect and the effect is mediated by inhibition of fat gain.

• Journal of Periodontal and Implant Science
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• v.50 no.5
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• pp.281-290
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• 2020

Purpose: The aim of this study was to compare microRNA (miRNA) gene expression in saliva using miRNA polymerase chain reaction (PCR) arrays in healthy and aggressive periodontitis (AP) patients. Methods: PCR arrays of 84 miRNAs related to the human inflammatory response and autoimmunity from the saliva samples of 4 patients with AP and 4 healthy controls were performed. The functions and diseases related to the miRNAs were obtained using TAM 2.0. Experimentally validated targets of differentially expressed miRNAs were obtained from mirTarBase. Gene ontology terms and pathways were analyzed using ConsensusPathDB. Results: Four downregulated miRNAs (hsa-let-7a-5p, hsa-let-7f-5p, hsa-miR-181b-5p, and hsa-miR-23b-3p) were identified in patients with AP. These miRNAs are associated with cell death and innate immunity, and they target genes associated with osteoclast development and function. Conclusions: This study is the first analysis of miRNAs in the saliva of patients with AP. Identifying discriminatory human salivary miRNA biomarkers reflective of periodontal disease in a non-invasive screening assay is crucial for the development of salivary diagnostics. These data provide a first step towards the discovery of key salivary miRNA biomarkers for AP.