• The Korean Journal of Mycology
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• v.42 no.3
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• pp.219-224
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• 2014

A gram-negative bacterium was isolated from spent substrate of Agaricus bisporus and showed marked antagonistic activity against Pseudomonas tolaasii. The bacterium was identified as Pseudomonas azotoformans by based on the cultural, biochemical and physiological characteristics, and 16S rRNA gene sequence. The isolated bacterium was saprophytic but not parasitic nor pathogenic to cultivation mushroom. The isolated bacterium for P. tolaasii cell was not sufficient for inhibition in vitro. Control efficacy of Pseudomonas azotoformans HC5 to brown blotch of P. tolaasii was 73, 78, and 71% on A. bisporus, Flammulina velutipes, and Pleurotus ostreatus, respectively. In the future, the suppressive bacterium may be useful for development of a biocontrol system.

• Microbiology and Biotechnology Letters
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• v.26 no.3
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• pp.238-243
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• 1998

For simple and rapid detection of Pseudomonas tolaasii (PT), a bacterial brown blotch pathogen of oyster mushroom, enzyme-linked immunosorbent assays (ELISA) were developed. To produce specific antibody, PT ($5{\times}10^7$ cfu) and Freund's adjuvant were subcutaneously immunized into rabbits several times. By using the antiserum showing the highest titer, we established noncompetitive and competitive ELISA's. Standard curves of the ELISA's showed that the detection limits were $2{\times}10^2$cfu/ml and $3{\times}10^2$cfu/ml, respectively When investigated by noncompetitive ELISA, cross reactivities of the anti-PT antibodies against P. agarici, P. reactans, and other fluorescent Pseudomonas spp. were very low (<1/10$^3$), but those against P. solanacearum, Erwinia chrysanthemi, Streptococcus mutans, Xanthomonas citri, and a fungus Fusarium oxysporum were almost none. However, when investigated by competitive ELISA, the reactivities against any other strains except PT were almost none. When the ELISA's were applied to 18 strains derived from mushrooms in order to identify PT, only 11 strains showing both pathogenicity and white line reactivity were obviously positive. These results showed that the ELISA's could be convenient tools to detect PT in accordance with existing methods.

• Journal of Microbiology and Biotechnology
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• v.21 no.10
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• pp.1097-1100
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• 2011

A toxin produced by Pseudomonas tolaasii, tolaasin, causes brown blotch disease in mushrooms. Tolaasin forms pores on the cellular membrane and destroys cell structure. Inhibiting the ability of tolaasin to form ion channels may be an effective method to protect against attack by tolaasin. However, it is first necessary to elucidate the three-dimensional structure of the ion channels formed by tolaasin. In this study, the structure of the tolaasin ion channel was determined in silico based on data obtained from nuclear magnetic resonance experiments.

• Korean Journal of Ichthyology
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• v.20 no.1
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• pp.74-77
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• 2008

Three juvenile specimens (38.8~51.5 mm SL) of the opistognathid fish, Opistognathus hongkongiensis Chan were collected off the Jeju Island, southern Korea. O. hongkongiensis is characterized by having a large mouth, dark brown vertical bands on sides, and yellow dorsal fin without a black blotch. We described it as the first record to Korean fish fauna, and proposed the new Korean name, "Jul-hu-ak-chi" for this species.

• BMB Reports
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• v.40 no.1
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• pp.113-118
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• 2007

Tolaasin, a pore-forming peptide toxin, is produced by Pseudomonas tolaasii and causes brown blotch disease of the cultivated mushrooms. P. tolaasii 6264 was isolated from the oyster mushroom damaged by the disease in Korean. In order to isolate tolaasin molecules, the supernatant of bacterial culture was harvested at the stationary phase of growth. Tolaasin was prepared by ammonium sulfate precipitation and three steps of chromatograpies, including a gel permeation and two ion exchange chromatographies. Specific hemolytic activity of tolaasin was increased from 1.7 to 162.0 HU $mg^{-1}$ protein, a 98-fold increase, and the purification yield was 16.3%. Tolaasin preparation obtained at each purification step was analyzed by HPLC and SDS-PAGE. Two major peptides were detected from all chromatographic preparations. Their molecular masses were analyzed by MALDI-TOF mass spectrometry and they were identified as tolaasin I and tolaasin II. These results demonstrate that the method used in this study is simple, time-saving, and successful for the preparation of tolaasin.

• Applied Biological Chemistry
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• v.50 no.4
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• pp.257-261
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• 2007

Tolaasin, a peptide toxin produced by Pseudomonas tolaasii, causes a serious disease on the cultivated mushrooms, known as brown blotch disease. Hemolysis using red blood cells was designed to measure the cytotoxicity of tolaasin molecules. Since tolaasin has two amine groups near the C-terminus, its membrane binding will be dependent on the ionic states of the amine groups. When the tolaasin peptide was titrated, its titration curve indicated the presence of titratable amine(s) at pH ranges from 7.0 to 9.6. When the pH-dependence of tolaasin-induced hemolysis was measured at various pHs, hemolysis was more efficient at alkaline pHs. In order to measure the membrane binding activity of tolaasin at different pHs, RBCs were incubated with tolaasin molecules for short time periods and washed out with fresh buffer. Because of the tolaasin binding during the preincubation period, fast hemolyses were observed at pH 8 or higher. These results imply that non-charged or less positively charged states of tolaasin molecules easily bind to membrane and show high hemolytic activity.

• The Korean Journal of Mycology
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• v.44 no.3
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• pp.171-175
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• 2016

A gram-negative bacterium was isolated from spent substrates of Agaricus bisporus and showed significant antagonistic activity against Pseudomonas agarici. The bacterium was identified as Alcaligenes sp. based on cultural, biochemical, physiological characteristics and a 16S rRNA sequence analysis. The isolate is saprophytic, but not parasitic or pathogenic on cultivated mushroom, whereas it showed strong inhibitory effects against P. agarici cells in vitro. The control efficacy of Alcaligenes sp. HC12 against brown blotch of P. agarici was up to 63% on Agaricus bisporus. The suppressive bacterium may be useful for the development of biocontrol systems.

• Journal of Applied Biological Chemistry
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• v.64 no.4
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• pp.369-374
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• 2021

Brown blotch disease of oyster mushrooms is caused by tolaasin and its analog peptide toxins which are produced by Pseudomonas tolaasii. Tolaasin peptides form pores in the plasma membrane and destroy the fruiting body structure of mushroom. Lysis of red blood cells, hemolysis, can be occurred by cytotoxic activity of tolaasin. The hemolytic activity of tolaasin is inhibited by metal ions, such as Zn²⁺ and Ni²⁺. When Gadolinium ion was added, a biphasic effect was observed on tolaasin-induced hemolysis, an increase in hemolysis at submillimolar concentrations and an inhibition at millimolar concentrations. The mechanism of gadolinium ion-induced inhibition of tolaasin activity may not be similar to those of the inhibitions by other metal ions. Since gadolinium ion has been reported to change a lateral pressure of lipid membrane by binding to the negative charges of membrane lipids, it may not directly work on the tolaasin channel gating, but rather decrease the stability of tolaasin channel by increasing firmness of membrane.

• Journal of Mushroom
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• v.17 no.1
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• pp.19-23
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• 2019

A gram-positive bacterium was isolated from the spent substrate of Agaricus bisporus that showed a marked antagonistic activity against Pseudomonas agarici. It was identified as Bacillus safensis HC42 based on its cultural, biochemical, and physiological characteristics, and 16S rRNA sequence. B. safensis HC42 was saprophytic, but not parasitic or pathogenic, in cultivated mushrooms and showed strong inhibition of P. agarici in vitro. Moreover, it showed a control efficacy of 66 % against browning disease caused by P. agarici in Agaricus bisporus. Therefore, B. safensis HC42 may be useful in the future for the development of a biocontrol system.

• Journal of Applied Biological Chemistry
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• v.62 no.3
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• pp.287-292
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• 2019

Pseudomonas tolaasii causes brown blotch disease on the oyster mushroom (Pleurotus ostreatus). Various pathogenic strains of P. tolaasii were isolated and divided into three subtypes, $P1{\alpha}$, $P1{\beta}$, and $P1{\gamma}$. For phage therapy, bacteriophages against to these subtype strains were applied to mushroom cultivation and very successful to prevent from the disease. In this study, bacteriophages were isolated against the representative strains of subtype pathogens and their polyclonal antibodies were synthesized to investigate structural relationship among capsid proteins of phages. Phage preparations over $10^{10}pfu/mL$ were injected to rabbit thigh muscle and polyclonal antibodies were obtained after three times of boost injection. Titers of the antibodies obtained were over $2{\times}10^7Ab/mL$ for the phage ${\phi}6264$, $1{\times}10^6Ab/mL$ for the phage ${\phi}HK2$, and $1{\times}10^7Ab/mL$ for the phage ${\phi}HK19$ and phage ${\phi}HK23$. High specific activities were observed between antibodies and the corresponding bacteriophages. Some cross-reactivities between the antibodies and non-corresponding bacteriophages were also measured. Antibody $Ab{\phi}6264$ inactivated all phages of $P1{\alpha}$ subtype and only phage ${\phi}HK16$ among $P1{\beta}$ subtype phages. Antibody $Ab{\phi}HK23$ of $P1{\gamma}$ subtype neutralized all phages of $P1{\beta}$ subtype as well as the phage ${\phi}HK23$, showing the widest phage-inactivation range. When the structural-similarity studies of phages were investigated by using phage antibodies, closeness obtained by phylogenetic analysis of 16S rRNA genes of pathogenic strains were quite different from that of polyclonal antibody-specific structural similarity of phage capsid proteins. In conclusion, there is weak correlation between the host strain specificity of bacteriophage and its capsid structural similarity measured by phage antibodies.