• Applied Biological Chemistry
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• v.50 no.2
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• pp.95-100
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• 2007

The cellulase gene of Bacillus licheniformis K11 which has plant growth-promoting activity by auxin and antagonistic ability by siderophore was cloned in pUC18 using PCR employing heterologous primers. The 1.6kb PCR fragment contained the full sequence of the cellulase gene, denoted celW which has been reported to encode a 499 amino acid protein. Similarity search in protein data base revealed that the cellulase from B. licheniformis K11 was more than 97% identical in amino acid sequence to those of various Bacillus spp. The cellulase protein from B. licheniformis K11, overproduced in E. coli DH5${\alpha}$ by the lac promoter on the vector, had apparent molecular weight of 55 kDa upon CMC-SDS-PAGE analysis. The protein not only had enzymatic activity toward carboxymethyl-cellulose (CMC), but also was able to degrade insoluble cellulose, such as Avicel and filter paper (Whatman$^{\circledR}$ No. 1). In addition, the cellulase could degrade a fungal cell wall of Phytophthora capsici. Consequently B. licheniformis K11 was able to suppress the peperblight causing P. capsici by its cellulase. Biochemical analysis showed that the enzyme had a maximum activity at 60$^{\circ}C$ and pH 6.0. Also, the enzyme activity was activated by Co$^{2+}$ of Mn$^{2+}$ but inhibited by Fe$^{3+}$ or Hg$^{2+}$. Moreover, enzyme activity was not inhibited by SDS or sodium azide.

• Microbiology and Biotechnology Letters
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• v.20 no.4
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• pp.384-389
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• 1992

Bacillus sp. YBL-7 which had been isolated from ginseng root-rot suppressive soil was able to antagonize Fusarium solani causing ginseng root-rot by their antibiotic substance. In order to develop multifunctional antagonist on Bacillus sp. YBL-7 as a biocontrol agent against Fusarium salam', optimal conditions for protoplast transformation system of Bacillus sp.YBL-7 by the vector plasmid pE194 were investigated. The protoplasts of Bacillus sp. YBL-7 were obtained at best efficiency by treatment with 200${\mu}g$/ml of lysozyme in the pH 7.0 of SMM buffer for 90 minutes at $40^{\circ}C$. The cell wall of the protoplast was regenerated on the agar plate containing 1.2% agar and 0.7 M mannitol. Under the best condition for protoplast formation and regeneration, the optimal transformation was achieved with 40% polyethylene glycol (M.W. 4000) treatment for 10minutes. The vector plasmid pE194 showed the best transformation frequency at 5$\mu$g/ml of final concentration. The pE194 was very stable over 80% in the transformants.

• The Korean Journal of Mycology
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• v.24 no.2 s.77
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• pp.93-103
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• 1996

In order to find out effect on the inorganic and organic compounds as a soil amendment to control Fusarium wilt of cucumber caused by Fusarium oxysporum f. sp. cucumerinum, this study was conducted during the last two years from 1993 to 1995. According to add 14 inorganic chemicals (1%, w/v) including $Al_2(SO_4)_3$ individually in vitro, these chemicals were confirmed their suppression, and especially Alum, CaO and $Al_2(SO_4)_3$ suppressed not only $20.9{\sim}25.0$ percent on mycelial growth of the fungus, but also inhibited $72.8{\sim}97%$ on conidial germination. $Ca(NO_3)_2$ suppressed mycelial growth only, while KCl, $K_2SO_4,\;NH_4NO_3$, and Urea suppressed conidial germination. The 7 chemicals were finally seleced. Composted pine bark (CPB) suppressed definely more than 90% on conidial germination in the different extract concentration (2,5 and 10%), although mycelial growth on extract medium of CPB and milled alfalfa leaves (MAL) were not remarkable. The antagonist Trichoderma sp. (Tr-3) mixed with an amended soil (1%, w/w) containing composted pine bark showed a good mycelial growth to compete the causal fungus. And the antagonist Pseudomonas sp. (7-1-3) was also confirmed its antagonistic ability with culture filterate. It is known that a CPB soil amendment mixed with the two antagonists (1%, w/w) controlled almost completely Fusarium wilt of cucumber in greenhouse pots and a field experiment. It is therefore expected that biocontrol on Fusarium wilt of cucumber by a soil amendment can be applied to farmmer's fields.

• The Korean Journal of Mycology
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• v.42 no.3
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• pp.225-230
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• 2014

Sclerotinia sclerotiorum, a plant pathogenic fungus, can cause serious yield and quality losses in the winter lettuce field. For biological control of S. sclerotiorum, soil-born microorganisms that inhibit the mycelia growth of S. sclerotiorum and Fusarium oxysporum were isolated from diseased soil. Among the isolates, bacterial isolate, GG95, which was identified as Bacillus subtilis according to the morphological, physiological characteristics and by 16S rRNA similarity, showed the highest level of inhibitory activity. The growth conditions for B. subtilis GG95 were optimized in TSB media (pH 7) by culturing at $28^{\circ}C$ for 24 hrs. Maltose or fructose and peptone were selected as the best carbon and nitrogen sources, respectively. Greenhouse experiment was performed to test effectiveness of B. subtilis GG95 in the control sclerotinia rot. Drench application ($1{\times}10^8cfu/mL$, 3 times) of the bacterial culture broth to lettuce showed an effectiveness value of 88%, suggesting that B. subtilis GG95 would be a promising biocontrol agent for control of sclerotinia rot.

• Korean journal of applied entomology
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• v.43 no.3 s.136
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• pp.225-231
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• 2004

An endoparasitoid wasp, Cotesia plutellae, has been used for a biological control agent against the diamondback moth, Plutellae xylostella. It has a symbiotic polydnavirus in their reproductive tract, which is required for its successful parasitization. Here, we measured a specific replication time of the polydnavirus during female development of C. plutellae. We, also, analyzed the reproductive potentials of female C. plutellae under mating or different host conditions. At $25^{\circ}C$, pupal C. plutellae began to develop adult tissues such as compound eyes and wings since day 2. At day 5, all adult tissues including antennae were developed and were ready to emerge. With polyclonal antibody raised against C. plutellae polydnavirus, an immunobloting could confirm virus replication at day 4 during pupal stage. Virus particles could be visualized by transmission electron microscope in the oviduct lumen of day 5 pupae. After adult eclosion, venom gland and ovarian calyx increased in size, though ovarioles did not. Mated females layed large number of eggs (over $60\%$) at first 4 days during their mean longevity of ca. 8 days at $25^{\circ}C$. Unmated females showed less active ovipositional behavior, where all the eggs developed into males. C. Plutellae parasitized both P. xylostella and fall webworm, Hyphantria cunea. However, C. Plutellae developed faster and showed higher successful paarasitization in P. xylostella than in H. cunea.

• Research in Plant Disease
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• v.21 no.4
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• pp.280-289
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• 2015

Maize (Zea mays L.) is an economically important crop in worldwide. While the consumption of the maize is steadily increasing, the yield is decreasing due to continuous mono-cultivation and infection of soil-borne fungal pathogens such as Fusarium species. Recently, stalk rot disease in maize, caused by F. subglutinans and F. temperatum has been reported in Korea. In this study, we isolated bacterial isolates in rhizosphere soil of maize and subsequently tested for antagonistic activities against F. subglutinans and F. temperatum. A total of 1,357 bacterial strains were isolated from rhizosphere. Among them three bacterial isolates (GC02, GC07, GC08) were selected, based on antagonistic effects against Fusarium species. The isolates GC02 and GC07 were most efficient in inhibiting the mycelium growth of the pathogens. The three isolates GC02, GC07 and GC08 were identified as Bacillus methylotrophicus, B. amyloliquefaciens and B. thuringiensis using 16S rRNA sequence analysis, respectively. GC02 and GC07 bacterial suspensions were able to suppress over 80% conidial germination of the pathogens. GC02, GC07 and GC08 were capable of producing large quantities of protease enzymes, whereas the isolates GC07 and GC08 produced cellulase enzymes. The isolates GC02 and GC07 were more efficient in phosphate solubilization and siderophore production than GC08. Analysis of disease suppression revealed that GC07 was most effective in suppressing the disease development of stalk rot. It was also found that B. methylotrophicus GC02 and B. amyloliquefaciens GC07 have an ability to inhibit the growth of other plant pathogenic fungi. This study indicated B. methylotrophicus GC02 and B. amyloliquefaciens GC07 has potential for being used for the development of a biological control agent.

• Journal of Mushroom
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• v.22 no.2
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• pp.60-66
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• 2024

This study was conducted to selection and investigate appropriate conditions for mass production of antagonistic microbes to control cobweb disease caused by Cladobotryum mycophilum. A grampositive bacterium was isolated from spent substrate of Agaricus bisporus and showed significant antagonistic activity against Cladobotryum mycophilum. The bacterium was identified as Bacillus altitudinis. based on the cultural, biochemical and physiological characteristics, and 16S rRNA sequence. The isolate is saprophytic, but not parasitic nor pathogenic to cultivated mushroom whereas it showed strong inhibitory effects against C. mycophilum cells in vitro. The control efficacy of B. altitudinis HC7 against cobweb disease of C. mycophilum was up to 78.2% on Agaricus bisporus. The suppressive bacterium may be useful for the development of biocontrol system. To define the appropriate conditions for the mass production of the Bacillus altitudinis HC7, we have investigated appropriate culture conditions and effects of various nutrient source on the bacterial growth. The appropriate initial pH and temperature were determined as pH 6.0 and 30℃, respectively. The appropriate concentration of medium elements for the growth of pathogen inhibitor bacterium(Bacillus altitudinis HC7) was determined as follows: 3.0% soluble startch, 10% soytone, 1.0% (NH₄)₂HPO₄, 1.0 mmol KCl, and 0.5% L-asparagine.

• The Korean Journal of Pesticide Science
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• v.2 no.3
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• pp.52-63
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• 1998

A novel quinolineoxadiazoles, substituted the carboxylic acid group with 1,2,4-oxadiazole radicle, of KSC-16960 and related compounds were evaluated to examine the herbicidal activity, crop injury and residual effect of after-vegetable crops compared with those of quinclorac (3,7-dichloro-8-quinolinecarboxylic acid), of which use is now banned because of its residual activity to some succeeding vegetable crops. KSC-16960 showed 2- and 3-leaf stages of barnyardgrasses effectively controlled by more than 95 and 90%, respectively, at a rate of 6.25 g/ha. The dose of KSC-16960 controlled 4- and 5-leaf stages of barnyardgrasses by more than 90% were found to be 50 g and 100 g/ha, respectively. The selectivity of KSC-16960 between direct-seeded rice and barnyardgrass was approximately 2-fold higher than that of quinclorac when they were treated to the soil. The selectivity indices of KSC-16960 and of quinclorac between 1-leaf stage of direct seeded rice and 5-leaf stage of barnyardgrass were 44 and 23, respectively, and those between 1-leaf stage of direct seeded rice and 4-leaf stage of barnyardgrass were almost 2-fold higher. Application of KSC-16960 with bentazone exhibited an additive controlling effect on several weed species, but that of quinclorac exhibited an antagonistic effect. With pyrazosulfuron-ethyl, on the other hand, both application of KSC-16960 and quinclorac showed additive interactions. Under a greenhouse condition, the residual activity of KSC-16960 to succeeding tomato plants was approximately 4-fold lower compared to that of quinclorac. KSC-16960 could be substituted for quinclorac, if it will be made some more improvement for reducing residual activity.

• Korean Journal of Environmental Agriculture
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• v.24 no.4
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• pp.409-415
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• 2005

To control Phytophthora blight of red pepper biologically caused by Phytophthora capsici, we developed Trichoderma harzianum DYMC for commercial product. DYMC was storage at room temperature and was investigated their population every 3 months for 1 year. For investigating the dynamic population of T. harzianum in the pot soils, we applied powder and suspension applications with DYMC, and then investigated for 95 days. The efficacy of powder and suspension applications of DYMC for control of Phytophthora blight of red pepper and plant growth were investigated for 50 days in greenhouse experiment. The population of T. harzianum was decreased at the room temperature for 1 year but there was not statistically significance. After soil treated in the pot with DYMC, the population of Trichoderma spp. was the highest when DYMC powder at 5 g was applied to mix with pot soil, and the population was deceased significantly among treatment means as time goes by ($R^2=0.76$, F=10.5960, P=<.0001). Incidence of Phytophthora blight of, red pepper was significantly reduced among treatment means on 50th day after treated with DYMC ($R^2=0.82$, P=16.4758, P=<.0001). Disease control value was the highest at 62.5% when DYMC powder at 5 g was applied to mix with pot soil. No significant difference (P=0.05) of effects of plant and root growth showed by treated with DYMC on 60th day, except stem. Mixing the application of DYMC powder with soil to control Phytophthora blight of red pepper was greater than suspension application to dilute with water. DYMC could be used as an effective biocontrol agent to control Phythophthora blight of red pepper.

• Journal of Life Science
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• v.19 no.11
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• pp.1672-1678
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• 2009

The purified antifungal substances produced by Bacillus amyloliquefaciens IUB158-03 was positive to ninhydrin but negative to aniline, suggesting that the antifungal substance could be a peptide. FAB-MS, UV adsorption spectrum, and amino acid composition analysis revealed that the molecular weight of the antifungal substance was 1042 and that maximal adsorption was at 220 nm and 277 nm. The antifungal substance was composed of $Asn_3$, $Gln_2$, $Ser_1$, $Gly_1$, and $Tyr_1$. The composition and structural characteristics of antifungal substance were analysed by $^1H$-NMR spectrum, $^1H$-COSY, HMQC, which revealed that the compound belongs to the iturin A family. Temperature and pH had little effect on the stability of the antifungal substance in the ranges of $-70{\sim}121^{\circ}C$ and pH 6.0~10.0, respectively. It showed strong antibiotic activity against fungi. An in vitro cytotoxicity test using NIH3T3 cell showed that the antifungal substance does not have cytotoxicity. The number of circulating leukocytes and the hematobiological analysis of the mice administered with the antifungal substances was similar to those of the control group, indicating no cytotoxicity in vivo. Therefore, the antifungal substances extracted from culture broth of Bacillus amyloliquefaciens IUB158-03 have future potential as biocontrol agents against plant diseases caused by fungi.