• Korean Journal of Microbiology
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• v.38 no.2
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• pp.127-132
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• 2002

In this study, the bacterial communities distributed in sea water of the whitening areas of Gangjeong and Seongsan, Jeju-do have been analysed using the PCR amplification of 16S rRNA to obtain fundamental data and information on relationship of the whitening phenomenon and microbial ecosystem. In Gangjeong, diverse bacteria such as Alcanivorax, Paracoccus, Damselae, Pseudomonas, Rhodowlum, Silicibacter, Suiftobacter, and Roseobacter have been found, and Alcanivorax was the most abundant clone. The most abundant clone from Seongsan was Pseudomonas, of which Pseudomonas tolaasii and Pseudomonas mandeli were most abundantly occurred in the frequency of approx44% and 24%, respectively. Approx4% of the bacterial clones closest to Verrucomicrobiales and other unidentified clones were also fecund in Seongsan, suggesting there is a great discrepancy between bacterial communities from the whitening areas of Seongsan and Gangjeong. The mean temperature, chlorine concentration, pH, and dissolved oxygen (DO) of the sea water of Gangieong and Seongsan in August of 2001 (sampling period) was $27^{\circ}C$~$27.5^{\circ}C$, 30.24~30.60%, pH 8.23~8.36,7 .20~7.28 mg/ι, suggesting other environmental factors except far the factors mentioned above might result in difference of bacterial communities distributed in both areas.

• Applied Biological Chemistry
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• v.43 no.3
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• pp.190-195
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• 2000

Tolaasin is a peptide toxin produced by Pseudomonas tolaasii and causes a brown blotch disease forming brown, slightly sunken spots and blotches on the cultivated mushrooms. It is a lipodepsipeptide consisting of 18 amino acids and its molecular mass is 1,985 Da. It forms a pore in plasma membranes, resulting in the disruption of membranes of fungal, bacterial, plant, and animal cells as well as mushroom tissue. In order to measure the toxicity of tolaasin, erythrocytes of blood were used to evaluate the tolaasin-induced hemolysis. Hemolytic activity of tolaasin was measured by observing the absorbance change either at 420 nm, representing the release of hemoglobins from red blood cells(RBCs), or at 600 nm, representing the density of residual cells. The hemolytic activity of culture-extract of P. tolaasii increased at early-stationary phase of growth and was maximal at late stationary phase. The hemolytic activity of tolaasin appeared high in the RBCs of dog and rat. The RBCs of rabbit and hen were less susceptible to tolaasin. The effects of various cations were also measured. $Cd^{2+}$ and $La^{3+}$. as well as $Zn^{2+}$ appeared inhibitory to the tolaasin-induced hemolysis. The effects of various anions on tolaasin-induced hemolysis were measured and carbonate showed the greatest inhibition to the hemolysis. However, phosphate stimulated the tolaasin-induced hemolysis and no effects were observed by chloride and nitrate.

• Journal of Applied Biological Chemistry
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• v.59 no.3
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• pp.221-225
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• 2016

Tolaasin is a pore-forming peptide toxin produced by Pseudomonas tolaasii and causes a brown blotch disease by disrupting membrane structures of cultivated mushrooms. The mechanism and characteristics of tolaasin pore formation are not known in detail; however, tolaasin pores have been demonstrated in the artificial lipid bilayer. Since the tolaasin pore appeared less frequently and unstable in lipid bilayer, a mismatch between the length of tolaasin pore and the thickness of lipid membrane had been suggested. Therefore, tolaasin-induced hemolyses were measured by the additions of phospholipids composed of various fatty acids with different carbon numbers. When phosphatidylethanolamines made with two decanoic acids (C10:0, 1,2-didecanoyl-sn-glycero-3-phosphoethanolamine; DDPE), myristic acids (C14:0, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), and stearic acids (C18:0, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine) were added to the buffer containing RBCs and tolaasin peptides, DDPE facilitated the tolaasin-induced hemolysis while the other two phospholipids showed no effects. At various concentrations of DDPE, the tolaasin-induced hemolysis was stimulated as a dose-dependent manner. The phospholipids composed of mediumchain fatty acids stabilize the tolaasin pore probably by binding between the pore structure and membrane phospholipids and making the membrane thickness thinner around the pore. These results showed that tolaasin molecules make more stable pores in the membrane made with phospholipids composed of medium length fatty acids, suggesting that the length of tolaasin pore is a little shorter than the thickness of RBC membrane.

• Journal of Applied Biological Chemistry
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• v.60 no.2
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• pp.179-184
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• 2017

Tolaasin, a 1.9 kDa peptide toxin, is produced by Pseudomonas tolaasii and causes the brown blotch disease of cultivated oyster mushroom. It forms pores on the membrane and thus destroys cellular membrane structure, seriously reducing the productivity of mushroom cultivation. The mechanism of tolaasin-induced cytotoxicity is not known in detail. However, it has been reported to form a pore structure in the cytoplasmic membrane through the molecular multimerization. Therefore, food additives which can interact with tolaasin molecules may inhibit the pore formation by hydrophobic interactions with tolaasin molecules. In this study, various food additive materials have been identified as inhibitors of the tolaasin activity and named tolaasin-inhibitory factors (TIF). Most of TIFs are emulsifying agents for food processing procedures. Among various TIFs, polyglycerol and sucrose esters of fatty acids blocked effectively the cytotoxicity of tolaasins at the concentrations $10^{-4}-10^{-5}M$. These TIFs also successfully suppressed the blotch disease development in the shelf cultivation of oyster mushroom.

• Applied Biological Chemistry
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• v.49 no.4
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• pp.281-286
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• 2006

Tolaasin, a pore-forming toxin, is a 1,985 Da peptide produced by Pseudomonas tolaasii and causes a brown blotch disease on cultivated mushrooms. Tolaasin forms pores on the plasma membrane of various cells including fungi, bacteria, plant as well as erythrocytes, and destroys cell structure. $Zn^{+2}$ has been known to block the tolaasin activity by an unknown mechanism. Thus, we investigated the inhibitory effects of $Zn^{+2}$ on the tolaasin-induced hemolysis to understand the molecular mechanism of tolaasin-induced pore formation. $Zn^{+2}$ and $Cd^{+2}$ inhibited the tolaasin-induced hemolysis in a dose-dependent manner and their Ki values were 170 ${\mu}M$ and 20 mM, respectively. The effect of $Zn^{+2}$ was reversible since the subsequent addition of EDTA chelates $Zn^{+2}$ and removes the inhibitory effect of $Zn^{+2}$. When an osmotic protectant, PEG 2000, was added, the tolaasin-induced hemolysis was not observed. After the removal of osmotic protectant by centrifugation, resuspended erythrocytes with fresh medium were immediately hemolyzed, while the addition of $Zn^{+2}$ prevented from hemolysis, implying that tolaasin-induced pores on the membrane were already formed in the medium containing osmotic protectant. These results suggest that $Zn^{+2}$ inhibits the activity of tolaasin pores and it has minor effects on the membrane binding of tolaasin and the formation of pore.

• Journal of Applied Biological Chemistry
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• v.60 no.4
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• pp.351-355
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• 2017

Tolaasin, peptide toxin produced by Pseudomonas tolaasii, causes a brown blotch disease on the cultivated mushrooms. Tolaasin peptides form membrane pores and disrupt cellular membrane structure. Molecular actions of tolaasin consist of the aggregation of peptide molecules, binding to the cell membrane, and formation of membrane pores. Therefore, the inhibitions of any of these actions are able to suppress the blotch disease. We have isolated and identified several tolaasin inhibitors (named tolaasin inhibitory factors, TIF) from food additives. TIFs were able to suppress the blotch-formation by the pathogen inoculated to the mushrooms. In this study, TIFs were incubated under various conditions and their activities for the inhibition of tolaasin-induced hemolytic activity were investigated. Since TIFs are unsaturated carbon compounds, they were sensitive to the air exposure and light irradiation. In the anaerobic conditions, TIFs were stable and their activities were decreased by 10% for three months. However, near 90% of TIF activity was suppressed by two weeks in the presence of air and sun light. Temperature did not show any significant effects on the activity of TIF, since storages at 5, 25, $45^{\circ}C$ did not show any difference. Therefore, for the stable storage of TIF compounds, container should be designed to be dark and air-tight.

• The Korean Journal of Mycology
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• v.38 no.1
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• pp.69-74
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• 2010

The 2 strains of Pleurotus ostreatus showing very severe and mild symptom of brown blotch were selected, and their F1 hybrid strains were made by mating of their single spore isolates. Hydrophobicity of fruiting body surface and brown blotch severity of the parental and their 11 F1 hybrid strains showing different level of severity of brown blotch were determined. Correlation coefficient (R) between disease severity and hydrophobicity were 0.68 and 0.70 on two independent experiments. Correlation coefficient between disease severity and disease incidence that was determined in the oyster mushroom farm were 0.46 and 0.52 on two independent experiments. When GFP-tagged Pseudomonas tolaasii was monitored with confocal microscope on cap surface of fruiting body, more cells of the pathogen were clustered on the severe strain than the mild strain, which indicates that the bacterial pathogen proliferates more on the severe strain. These results suggest that hydrophobicity of oyster mushroom fruiting body affects disease severity of the brown blotch disease; the longer the bacterial suspension stays on fruiting body surface more severe symptom of the blotch develops.

• Journal of Mushroom
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• v.22 no.2
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• pp.67-72
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• 2024

In this study, we investigated the microbial community of oyster mushrooms at different growth stages at the species level. Gram-positive bacteria were predominant in the presterilized medium. On the other hand, Gram-negative bacteria were predominant in the culture-completed medium, post-harvest medium, and fruiting bodies. In addition, Pseudomonas tolaasii, which is known to cause disease in mushrooms, was confirmed in the cultured medium, post-harvest medium, and fruiting bodies, and it was determined that the mycelium culture stage was contaminated, and the reason why no disease occurred was Sphingobacterium psychroaquaticum. It was confirmed that this was because the growth of Pseudomonas tolaasii was suppressed by producing a component called tolacin. As a result of confirming the diversity of microorganisms, it was confirmed that the presterilization medium contains a variety of microorganisms compared to other growth stages, and the diversity decreases in the order of culture completion medium, fruiting body, and post-harvest medium. showed a trend. As a result of microbial similarity analysis, it was confirmed that the cultured medium and the post-harvest medium showed similar microbial communities, and in the case of fruiting bodies, there were some similarities but overall differences.

• The Korean Journal of Mycology
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• v.45 no.4
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• pp.370-376
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• 2017

Pseudomonads cause bacterial brown blotch disease, which causes great damage to the common mushroom Agaricus bisporus. The tolerance of A. bisporus to pseudomonads was tested and found to not be correlated with mycelium growth ability. The offsprings of the tolerant strain (ASI1085) to pseudomonads were not as tolerant as their parents in the mycelium stage. But, tolerance decreased compared to mycelium in the fruiting body. The offsprings of the weakly tolerant strain (ASI1321) were even more weak in the mycelium stage. It is presumed that the tolerance of the parents is transferred to later generations. The tolerance in the mycelium was not correlated in the fruiting body. Therefore, the browning of the fruiting body is thought to be induced by other factors. Pseudomonas tolaasii caused higher browning than Pseudomonas agarici. Pseudomonas reactans did not have a significant effect on the mycelium, but affected the browning of the fruit bodies. P. agarici had higher ability to inhibit mycelium growth than fruiting body growth.

• Proceedings of the Korean Biophysical Society Conference
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• 1998.06a
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• pp.34-34
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• 1998

Tolaasin is a channel forming bacterial toxin produced by Pseudomonas tolaasii and causes a brown blotch disease on cultivated oyster mushrooms. When tolaasin molecules form channels in the membranes of mushroom cells, they destroy cellular membrane structure, known as 'colloid osmotic lysis'. In order to understand the molecular mechanisms forming membrane channels by tolaasin molecules, we have investigated the electrophysiological characteristics of tolaasin-induced channels in lipid bilayer.(omitted)