• 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.

• 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.

• The Korean Journal of Mycology
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• v.41 no.4
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• pp.248-254
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• 2013

A Gram-negative bacterium was isolated from mushroom media that markedly reduces the level of extracellular toxins (i.e., tolaasins) produced by Pseudomonas tolaasii, the most destructive pathogen of cultivated mushrooms. The HC1 strain was selected as detoxifying tolaasin by bioassay on potato and it was identified Pseudomonas sp. by the cultural, morphological and physiological characteristics, and analysis of the 16S rRNA. The isolated bacterium is saprophytic but not parasitic nor pathogenic to cultivation mushroom. The isolated bacterium for P. tolaasii cell, was sufficient for detoxification in vitro. Inoculation of the isolated bacterium prevents the development of bacterial disease in Pleurotus ostreatus, Flammunia velutipes and Agaricus bisporus. Control efficacy of brown blotch of strain HC1 treatment was 69, 68 and 55% on Agaricus bisporus, Flammulina velutipes and Pleurotus ostreatus, respectively. The suppressive bacterium may be useful in future for the development of biocontrol system and the construction of genetically modified edible fungi resistant to the disease caused by P. tolaasii.

• Journal of Mushroom
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• v.9 no.4
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• pp.135-138
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• 2011

'Gonji-2ho' a new variety of oyster mushroom, fitting for the bag culture, was bred by mating between monokaryons isolated from GMPO35338 and Jangpug. In the major characteristics of fruit body, the pilei were thick and dark-gray and the stipes were thick and long with softness. It was great in elasticity and cohesivness of tissue as compared to Suhan-1ho. The optimum temperature for the mycelial growth was around $26{\sim}29^{\circ}C$ and for the pinheading and growth of fruit body was around $14{\sim}18^{\circ}C$. In the bag culture, it was required around 20 days at incubation period and 5 days at primordia formation. The fruit body was grown vital and uniform. The yields were 323.3g/kg bag. This variety has high yielding capacity, cultivation stability and the resistance to the bacterial brown blotch disease.

• 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.61 no.3
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• pp.213-217
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• 2018

Tolaasin secreted by Pseudomonas tolaasii is a peptide toxin and causes brown blotch disease on the cultivated mushrooms by collapsing cellular and fruiting body structure. Toxicity of tolaasin was evaluated by measuring hemolytic activity because tolaasin molecules form membrane pores on the red blood cells and destroy cell membrane structure. In the previous studies, we found that tolaasin cytotoxicity was suppressed by $Zn^{2+}$ and $Ni^{2+}$. $Ni^{2+}$ inhibited the tolaasin-induced hemolysis in a dose-dependent manner and its $K_i$ value was 1.8 mM. The hemolytic activity was completely inhibited at the concentration higher than 10 mM. The inhibitory effect of $Zn^{2+}$ on tolaasin-induced hemolysis was increased in alkaline pH, while that of $Ni^{2+}$was not much dependent on pH. When the pH of buffer solution was increased from pH 7 to pH 9, the time for 50% hemolysis ($T_{50}$) was increased greatly by $100{\mu}M$ $Zn^{2+}$; however, it was slightly increased by 1 mM $Ni^{2+}$ at all pH values. When the synergistic effect of $Zn^{2+}$ and $Ni^{2+}$ on tolaasin-induced hemolysis was measured, it was not dependent on the pH of buffer solution. Molecular elucidation of the difference in pH-dependence of these two metal ions may contribute to understand the mechanism of tolaasin pore formation and cytotoxicity.

• Korean Journal of Breeding Science
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• v.40 no.2
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• pp.173-177
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• 2008

A new peanut variety "Pungsan" (Arachis hypogaea ssp. hypogaea L.) was developed at the Yeongnam Agricultural Research Institute, NICS, in Milyang in 2007. It was derived from a cross between a short stem cultivar "Satonoka" and a high-yielding local cultivar "Yecheon". "Pungsan" is the Virginia plant type. It has 18 branches per plant and 40 cm of main stem height. Each pod with long-ellipse shaped large kernel has two grains with light-brown testa and 100 seed weight was 95g in the regional yield trials (RYT). "Pungsan" showed more resistant to web blotch compared with check variety. In the regional yield trials "Pungsan" was outyielded in grain yield to the check variety by 16% with 4.45 MT/ha for grain.