• 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.

• Korean Journal Plant Pathology
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• v.14 no.2
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• pp.177-183
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• 1998

A DNA fragment which is involved in tolassin production was cloned to obtain a molecular marker of Pseudomonas tolaasii, a casual agent of bacterial brown blotch disease of oyster mushroom (Pleurotus ostreatus). Tolaasin is a lipodepsipeptide toxin and known as a primary disease determinant of the P. tolaasii. It is responsible for formation of white line in agar when P. tolaasii were cultured against white line reacting organisms (WLROs). White line negative mutants (WL-) were generated by conjugation between rifampicin resistant strain of P. tolaasii and E. coli carrying suicidal plasmid pSUP2021 : : Tn5. The ability of tolaasin production of the WL- mutants was examined by hemolysis test, pathogenicity test, and high pressure liquid chromatography (HPLC) analysis of culture filtrate. All of the WL- mutants were lost the ability of tolaasin production (Tol-). Genomic library of the Tol- mutant was constructed in pLAFR3 and the cosmid clone containing Tn5 was selected. DNA fragment fro franking region of Tn5 was cloned from the plasmid and used as a probe in Southern blot. DNA-DNA hybridization with the probe to total DNA from group of bacteria ecologically similar to P. tolaasii including WLORs, fluorescent Pseudomonads isolated from oyster mushroom, P. agarici, P. gingeri, and some of other species of Psedomonas showed that some of the tested bacteria do not have any hybridized band and others have bands sowing RFLP. The cloned DNA fragment or its nucleotide sequence will be useful in detection and identification of the P. tolaasii.

• 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.

• Journal of Mushroom
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• v.1 no.1
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• pp.15-20
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• 2003

Antibacterial activities of 33 antibiotics against Pseudomonas tolaasii causing the brown blotch disease on the edible mushroom Pleurotus ostreatus, were tested in vitro for the control of the disease. Tetracyclin, kanamycin, kasugamycin, and streptomycin showed strong antibacterial activity against P. tolaasii, having the minimal inhibitory concentration of 10, 10, 100 and 200ppm, respectively. These antibiotics showed similar control value of 72.9, 71.2, 68.1 and 62.7%, respectively when applied on the artificially infected mushroom beds. Mushroom yields in the tetracycline treated boxes were increased about 31.8% comparing to the control ones. Mycelial growth of P. ostreatus on the PDA supplemented with streptomycin and kanamycin were not affected, but were inhibited 10~20% and 40% with tetracyclin and kasugamycin treatment, respectively.

• The Korean Journal of Mycology
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• v.22 no.2
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• pp.190-195
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• 1994

Studies were conducted to determine the potential of sodium hypochlorite(SHC) on the control of bacterial yellow blotch in cultivated oyster mushroom, Pleurotus ostreatus. SHC at the concentration of 80 ppm was effective on the control of Pseudomonas agarici causing yellow blotch in oyster mushroom except number 916 isolate. In vitro the mycelial growth was slightly inhibited at the concentration higher than 100 ppm of sodium hypochlorite, but retardation of the mycelial growth was soon recovered. Spray of SHC solution at the concentration of 40-50 ppm per day significantly reduced the incidence of the yellow blotch without impairing the growth of oyster mushroom in field culture. However, the higher concentration of SHC(67 ppm) induced yellow brown or dark gray in color and deformed cap and elongated stripe in morphology of fruiting body. Results indicate that periodical spray of sodium hypochlorite seems to be the recommendable method for protection against bacterial yellow blotch disease in oyster mushroom without reducing food quality.

• Journal of Applied Biological Chemistry
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• v.61 no.4
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• pp.405-410
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• 2018

Pseudomonas tolaasii strain No. 6264 has been isolated from mushroom tissue and identified as one of the major pathogen causing brown blotch disease. It secretes peptide toxins, known as tolaasin and its analogue peptides. P. tolaasii 6264 has been used as a typical pathogenic strain to study the brown blotch disease for last 20 years after confirming its blotch-forming ability, hemolytic activity, and white line formation. In this study, the characteristics of P. tolaasii 6264 strain were analyzed and compared according to storage period. Strains of P. tolaasii 6264 stored annually since 2012 were cultured and their pathogenic characters were analyzed. When the 16S rRNA sequences were compared, all strains were divided into two groups. Pathogenic characters including hemolytic activity, blotch-forming ability, and white line test were also investigated. The strains, P. tolaasii 6264-15-2 and P. tolaasii 6264-17, had all three activities; however, the rest of stored strains showed only blotch-forming ability losing other pathogenic characters. Tolaasin peptides were purified from the bacterial cultures and analyzed by mass spectrometry. The strains, P. tolaasii 6264-15-2 and P. tolaasii 6264-17, secreted Tol I (1987 Da), Tol II (1943 Da), and its analogues (1973 Da, 2005 Da) while some of these peptides were not found in the media cultured other strains. These results indicate that the pathogenicity of P. tolaasii could be varied during the storage period.

• 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.

• 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)

• Proceedings of the Korean Biophysical Society Conference
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• 1999.06a
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• pp.47-47
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• 1999

Tolaasin is a bacterial paptide toxin which is produced by Pseudomonas tolaasii. It forms pores in the cellular membranes, causing the brown blotch disease on the cultivated oyster mushroom. Previously, we showed that tolaasin-induced pore formation required the multimerization of tolaasin molecules. In order to measure the ionic effect on the tolaasin multimerization, the time course of tolaasin-induced hemolysis was measured in the presence of various cations and anions.(omitted)

• Journal of Food Hygiene and Safety
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• v.16 no.3
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• pp.232-240
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• 2001

One hundred twenty five bacterial isolates were obtained from the brown blotch-diseased oyster mushrooms collected from markets. Among them, 45 were determined as pathogenic bacteria and white line forming organisms(WLFO) were 6 strains and white line reaction organisms (WLRO) were 6 strains. All of the white line forming isolates were identified as Pseudomonas tolaasii which is a known pathogen of brown blotch disease of oyster mushroom by GC-MIS(Gas chromatography-microbial identification system). Six of the white line reacting organisms were identified as P. chlomraphis, P. fluorescens biotype A and type C. The rest of them were P gingeri, P. agarici, P. fluorescens biotype B, P. chloroyaphis, non-pathogenic P. tolaasii, P. putida biotype A and B etc. For spectrum of activity of tolaasin, culture filtrates from pathogenic isolates were examined by browning of mushroom tissue and pitting of mushroom caps. The weak pathogenic bacteria didn't induce browning or pitting of mushroom tissue. On the other hand, strong pathogenic isolates showed browning and pitting reaction on mushroom. An extracellular toxin produced by P. tolaasii, was investigated. The hemolysis activity test of 6 strains identified as P. tolaasii were 0.8∼0.9 at 600 nm and 3 strains of WLRO were 0.9∼1.0 and Pseudomonas app. were 1.0∼1.2. Observation of fresh mushroom tissue using confocal laser scanning microscopy was carried out for images of optical sectioning and vertical sectioning. Also images of brown blotch diseased oyster mushroom tissue after contamination P. tolaasii was obtained by CLSM.