• Applied Biological Chemistry
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• v.38 no.5
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• pp.435-439
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• 1995

Some characteristics and pathogenicity of Hyphantria cunea nuclear polyhedrosis virus (HcNPV), a potential microbial pesticide was studied. H. cunea NPV replicated in the nucleus of S. frugiperda cells cultured in the TNMFH medium. In case of virus infected cell, prepolyhedra formation was observed at 24hrs post-infection. At 48 hrs post-infection, Most of the infected cell contained many mature polyhedra which were released into culture media 72 hrs post-infection, with the cells grown in suspension culture, pH of the culture medium increased during the virus replication: the pH of fresh medium was 6.35 and rose to 6.77 within 120 hrs. Polyhedra formed a band in linear density gradient of sucrose by centrifugation, which co-sedimented with $50{\sim}55%$ sucrose. The shape of the purified polyhedra was mostly tetragonal hexahedron and its size was about $2.5{\mu}m$. Electron microscopy and phase contrast microscopy showed that many bundled nucleocapsids were occluded in mature polyhedra at 48 hrs post infection. H. cunea larvae infected with NPV showed a higher motality in the second and third instar than in the fourth instar. Death rate of H. cunea larvae in the second and third instar fed with leaves coated with $1.5{\times}10^{9}{\sim}l.5{\times}10^{7}PIBs/ml$ reached more than 90%.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2018.04a
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• pp.45-45
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• 2018

We had already reported the successful germination for green pods of purple lady's slipper orchid (Cypripedium macranthos Sw.). The green pod methods is to take immature seeds in green capsules, sterilize the capsule, and take out the sterile seeds. This method, however, needs very critical time of harvest. The critical time of seed harvest changes depending upon the species, condition of the specimen, and climatic influence, and the right time lies between 5 and 12 weeks after fertilization. In this study, the mature seeds were collected after 120-130 days with hand-polination of lady's slipper orchids. Mature seeds are usually dormant and it has to be overcome, either with hormone or storing the seeds near freezing for two or three months to break dormancy. The seeds were first surface sterilized with 70% ethanol and then transferred 1% NaOCl for 10-15 minutes, followed by rinses 3 times with sterilized distilled water. The cypripedium seeds consists of an embryo within a seed coat known as a testa. The testa is water repellent and the seed has a large air space between the embryo and testa so the seed tends to float on water. We had resolved the problems with vacuum pump to soak water into the testa before sterilization. The seeds were placed on liquid or agar solidified germination media. Cultures were incubated at $24{\pm}1^{\circ}C$ in dark. The seeds were germinated in 6-8 weeks in liquid suspension culture (germination percentage over 18%); however, the seeds on agar solidified media took more than 5 months to germinate and the germination percentage less than 5%. The most effective media for liquid culture was 1/4 strength Murashige and Skoog (MS) medium with 50 ml/l coconut water ($4brix^{\circ}$) at pH 5.8.

• Korean journal of applied entomology
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• v.56 no.4
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• pp.403-412
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• 2017

To investigate the control effect of insect pests by natural enemies, sticky traps were used in commercial tomato greenhouses in Chun-cheon and the experiment fields of Gangwon-do Agricultural Research and Extension Services, Republic of Korea using semi-forcing culture (February to June). We selected low toxicity pesticides, environment-friendly agricultural materials (EFAM), and natural enemies of the study species, combinations of which have been previously used in farms to control insect pests. In this study, Trialeurodes vaporariorum and thrips, which are major agricultural insect pests, were studied in experimental greenhouses. The adult T. vaporariorum population was observed in mid-April and the population of thrips showed occurrences in early April. Regarding seasonal fluctuation, T. vaporariorum peaked in mid-May and thrips peaked after June. one insecticide, spiromesifen suspension concentrate (SC); one fungicide, cyazofamid SC; and two EFAMs containing neem tree extract were shown to be slightly toxic to Encarsia formosa and Orius laevigatus. The results of this study could be used to develop management models using natural enemies of control the insect pests; T. vaporariorum and thrips in semi-forcing culture of tomato.

• Korean Journal of Plant Tissue Culture
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• v.24 no.1
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• pp.57-61
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• 1997

We investigated changes in the superoxide dismutase (SOD) activity and SOD isoenzyme pattern in suspension cultures of tomato (Lycopersicon esculentum), which were compared with those of intact tomato plants. two grams (fr wt) of cells subcultured at 15-day intervals were inoculated into 50 mL MS medium containing l mg/L 2,4-D and 30 g/L sucrose in a 300 mL flask and maintained at $25^{\circ}C$ in the dark (100 rpm). The cell growth reached a maximum at 20 days after subculture (DAS), followed by a rapid decrease with further cultures. The cell colour changed from white to black from 23 DAS. The intracellular SOD activity (units/g cell dry wt) was significantly increased from 23 DAS and reached a maximum at 28 DAS (52,400 units), followed by a decrease with further cultures, whereas the extracellular SOD activity showed a maximum at 25 DAS (27,800 units/50 mL medium). The total SOD activity per flask showed a maximum at 25 DAS (35,700 units), in which the extracellular SOD activity occupied about 75%. The tomato cultured cells had four SOD isoenzymes and their patterns were well correlated with SOD activity without a qualitative change during the cell cultures. The intact tomato plants had an additional CuZnSOD isoenzyme, showing the different isoenzyme patterns from cultured cells.

• Korean Journal of Plant Resources
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• v.30 no.5
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• pp.542-548
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• 2017

Two different races of Botryris cinerea were selected by the response of plant leaves to the pathogen infection. Based on lesion size of the pathogen on the leaves, turnip showed susceptible response to 'Grape-01' race, and resistant to 'Orange' race. Turnip leaves infected with resistant pathogen race, "Orange", showed significantly higher content of indole-3-ylmethyl glucosinolate (I3M) than those infected with susceptible race, 'Grape-01'. Contents of I3M in the leaves with resistant 'Orange' race was 2.5 times as high as that in uninfected leaves, whereas I3M in the leaves infected with susceptible 'Grape-01' race showed lower content than in untreated leaves. Growth of turnip suspension cells was significantly inhibited by the treatment of MeOH extract or water extract of 'Orange' race as compared with the treatment of susceptible race, 'Grape-01'. Treatment of MeOH or water extract from 'Orange' race to turnip suspension cells, strongly inhibited cell viability up to 22.7% or 16.5%, respectively. However, plant cells treated with MeOH or water extract from resistant race, 'Orange' showed higher I3M content than that from susceptible race, 'Grape-01'. These results suggest that accumulation and degradation of I3M glucosinolate in turnip cells closely related to the resistance and susceptibility of turnip cells to Botrytis cinerea.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.69-69
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• 2002

Transgenic animals production tools have been valuable for research and purpose. The current methods of gene transfer, microinjection and nuclear transfer, which are widely used in transgenic animal production, but all most methods has only had limited success in production of larger species. Here, we report the possibility of a sperm-mediated gene transfer method in porcine embryos. Oocytes were collected from ovaries harvested at a local slaughterhouse were matured in 500${mu}ell$ drops of TCM-199 under mineral oil at 38.5$^{\circ}C$ in a humidified atmosphere of 5%CO2 in air. After 42-43h of in vitro maturation oocytes were denuded. for sperm injection into the cytoplasm of the porcine oocytes, sperm suspension in NIM medium are subjected extraction with TritonX-100 before mixing with a green fluorescent gene (GFP). Sperm with Tritonx-100 were prepared by adding TritonX-100 to a final volume of 0.05% in the sperm suspension and mixing by trituration for 60s before two wishes in NIM medium at 2$^{\circ}C$. A(ter wishing, sperm were mixed with TritonX-100 at $25^{\circ}C$ followed by washes at 2$^{\circ}C$. Sperm were resuspended in ice cold NIM to a final volume of 400${mu}ell$ and 2-20ng/${mu}ell$ DNA were triturated on ice for 60s. All microinjection was performed in HEPES-buffered CZB medium at room temperature within 2h. After culture in NCSU-23 for 72h, percent of porcine embryos transfected GFP gene are 20.7%(6/29) in 20ng/${mu}ell$ sperm-DNA mixed group and other groups were 3.7 %(2/54)and 4.7%(3/67). These data suggests that sperm-mediated gene transfer method should be used to the production tool of transgenic pig efficiently.

• Korean Journal of Animal Reproduction
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• v.18 no.4
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• pp.235-244
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• 1995

The present study was designed to demonstrate that ES cell lines efficiently could be isolated from explanted blastocysts of hybrid BCF1 mouse when grown on STO feeder layer derived from mouse fibroblasts in culture medium supplemented with leukemia inhibitory factor (LIF). The expanded blastocysts were attached to mitomycin C-inactivated STO feeder layer and were cultured for 4 days. Four days later the ICM was disaggregated by a short term trypsin treatment (0.25% trypsin / 0.04% EDT A for 2-3 min). The resulting cell suspension was seeded on a new STO feeder layer and covered with DMEM supplemented with 10% FCS, 0.1 mM nonessential amino acid, 0.1 mM sodium pyruvate, 0.1 mM mercaptoethanol and 1,000 U/ml LIF. Colonies of ES-like cells were observed after the second passage. These colonies were repeatedly passaged at approximately 5 day intervals. In this study, five ES-like celllines were isolated by directly explanting blastocysts, but three lines were lost after the 5th passage, possibly due to toxic effects of a new FCS batch. The characterization of developmental potential of isolated cell lines was performed with respect to in vitro differentiation and specific activity of alkaline phosphatase (AP). When cells were cultured in suspension, the aggregates of cell lines were capable of forming simple embryoid bodies (EB), and showed the capacity for forming cystic multilayer EBs. In addition, the cell lines were positive for AP staining, a biochemical marker characteristic of mouse ES cells.

• Microbiology and Biotechnology Letters
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• v.20 no.5
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• pp.527-533
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• 1992

The optimal conditions for cellulase and xylanase production by Trichoderma reesei 28217 were studied for enzymatic deinking of old newspaper. The amounts of cellulase and xylanase from the strain was varied by initial medium pH, Tween 80, inoculum size of spore suspension, and carbon and nitrogen sources. The optimal conditions for cellulase production were pH 5.0-6.5, 0.02% of Tween 80, 0.5-1.0% of inoculum size of spore suspension ($1{\times}10^{7}$/ml). cottonseed meal as nitrogen source, and corn flour as carbon source. On the other hand, the optimal conditions for xylanase production were pH 6.5, 0.01% of Tween 80, corn steep liquor as nitrogen source, and disintegrated old newspaper as carbon source. The inoculum size for xylanase production was the same as for cellulase production. The concomitant production of cellulase and xylanase in shake flask culture was efficiently induced in the medium containing 0.5% cottonseed meal as nitrogen source and 1.0% old newspaper and 2.0% corn flour as carbon sources. In this case the activities of cellulase and xylanase produced were 6.11-7.22 IU/mJ and 97.7 IU/ml. respectively. However, the cellulase production in $5{\ell}$ fermentor scale was slightly decreased compared with that in flask scale. Moreover, xylanase production was severely reduced in a fermentor scale. The study for the reason of decreased enzyme production in fermentor is further needed.

• 한국균학회소식:학술대회논문집
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• 2014.10a
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• pp.34-34
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• 2014

Filamentous fungi of the genus Colletotrichum (teleomorph, Glomerella) are considered major plant pathogens worldwide. Cereals, legumes, vegetables, and fruit trees may be seriously affected by this pathogen (1). Colletotrichum species cause typical disease symptoms known as anthracnoses, characterized by sunken necrotic tissue, where orange conidial masses are produced. Anthracnose appears in both developing and mature plant tissues (2). We investigated disease occurrence in apple orchards from 2013 to 2014 in northern Gyeongbuk province, Korea. Typical anthracnose with advanced symptoms was observed in all apple orchards studied. Of late, static fruit spot symptoms are being observed in apple orchards. A small lesion, which does not expand further and remains static until the harvesting season, is observed at the beginning of fruit growth period. In our study, static symptoms, together with the typical symptoms, were observed on apples. The isolated fungus was tested for pathogenicity on cv. 'Fuji apple' (fully ripe fruits, unripe fruits, and cross-section of fruits) by inoculating the fruits with a conidial suspension ($10^5$ conidia/ml). In apple inoculated with typical anthracnose fungus, the anthracnose symptoms progressed, and dark lesions with salmon-colored masses of conidia were observed on fruit, which were also soft and sunken. However, in apple inoculated with fungi causing static symptoms, the size of the spots did not increase. Interestingly, the shape and size of the conidia and the shape of the appressoria of both types of fungi were found to be similar. The conidia of the two types of fungi were straight and cylindrical, with an obtuse apex. The culture and morphological characteristics of the conidia were similar to those of C. gloeosporioides (5). The conidia of C. gloeosporioides germinate and form appressoria in response to chemical signals such as host surface wax and the fruitripening hormone ethylene (3). In this study, the spores started to germinate 4 h after incubation with an ethephon suspension. Then, the germ tubes began to swell, and subsequently, differentiation into appressoria with dark thick walls was completed by 8 h. In advanced symptoms, fungal spores of virtually all the appressoria formed primary hyphae within 16 h. However, in the static-symptom fungus spores, no primary hyphae formed by 16 h. The two types of isolates exhibited different growth rates on medium containing apple pectin, Na polypectate, or glucose as the sole carbon. Static-symptom fungi had a >10% reduction in growth (apple pectin, 14.9%; Na polypectate, 27.7%; glucose, 10.4%). The fungal isolates were also genetically characterized by sequencing. ITS regions of rDNA, chitin synthase 1 (CHS1), actin (ACT), and ${\beta}$-tubulin (${\beta}t$) were amplified from isolates using primer pairs ITS 1 and ITS 4 (4), CHS-79F and CHS-354R, ACT-512F and ACT-783R, and T1 and ${\beta}t2$ (5), respectively. The resulting sequences showed 100% identity with sequences of C. gloeosporioides at KC493156, and the sequence of the ${\beta}$t gene showed 100% identity with C. gloeosporioides at JX009557.1. Therefore, sequence data from the four loci studied proves that the isolated pathogen is C. gloeosporioides. We also performed random amplified polymorphic DNA-PCR, which showed clearly differentiated subgroups of C. gloeosporioides genotypes. The clustering of these groups was highly related to the symptom types of the individual strains.

• Journal of Environmental Health Sciences
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• v.40 no.3
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• pp.255-263
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• 2014

Objectives: This study evaluated the fungicidal efficacy of a fumigant containing 20% ortho-phenylphenol against Trichophytone mentagrophytes (T. mentagrophytes), Candida albicans (C. albicans) and Aspergillus niger (A. niger). Methods: Five replicates of each carrier were contaminated by depositing 0.05 mL of each fungal suspension. After drying, two carriers without exposure to the fumigant and three carriers with exposure to the fumigant were left in a sealed room ($25m^3$) at $21{\pm}0.5^{\circ}C$ and $60{\pm}10%$ relative humidity for 15 hours. Immediately after removal from the test room, each carrier was transferred into recovery diluent and suspended, diluted and inoculated. After incubation, the numbers of each colony were counted, and the parameter values (N, T, d) were calculated. Results: The working culture suspension number (N value) of T. mentagrophytes, C. albicans and A. niger were $1.0{\times}10^8$, $1.2{\times}10^8$ and $5.7{\times}10^7CFU/mL$, respectively. All the colony numbers on the carriers exposed to the fumigant (n1, n2, n3) were higher than 0.5N1 (the number of fungal test suspensions by pour plate method), 0.5N2 (the number of fungal test suspensions by filter membrane method) and 0.5N1, respectively. In addition, all mean numbers of test strains recovered on the control-carriers (T value) were over $10^6CFU/mL$. For the fungicidal effect of the fumigant, all numbers of fungal reductions after exposure of the fumigant (d value) were 4 logCFU/mL. Conclusions: The present study showed that fumigant containing 20% ortho-phenylphenol has effective fungicidal activity against T. mentagrophytes, C. albicans and A. niger.