• Applied Biological Chemistry
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• v.37 no.5
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• pp.392-396
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• 1994

The toxic substance causing blown blotching on the mushroom cap was extracted and purified from the culture broth of P.tolaasii. Purification and identification of the toxic compound was carried out with the silica gel chromatography, mass spectrum and NMR and appeared to be an aminobenzene in amylamine group. The purified toxic substance showed UV spectrum at 234nm and melting point at $76^{\circ}C$, respectively.

• 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 Plant Pathology Journal
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• v.38 no.5
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• pp.472-481
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• 2022

Brown blotch disease, caused by Pseudomonas tolaasii, is one of the most serious diseases in mushroom cultivation, and its control remains an important issue. This study isolated and evaluated pathogen-specific bacteriophages for the biological control of the disease. In previous studies, 23 varieties of P. tolaasii were isolated from infected mushrooms with disease symptoms and classified into three subtypes, Ptα, Ptβ, and Ptγ, based on their 16S rRNA gene sequences analysis and pathogenic characters. In this study, 42 virulent bacteriophages were isolated against these pathogens and tested for their host range. Some phages could lyse more than two pathogens only within the corresponding subtype, and no phage exhibited a wide host range across different pathogen subtypes. To eliminate all pathogens of the Ptα, Ptβ, and Ptγ subtype, corresponding phages of one, six, and one strains were required, respectively. These phages were able to suppress the disease completely, as confirmed by the field-scale on-farm cultivation experiments. These results suggested that a cocktail of these eight phages is sufficient to control the disease induced by all 23 P. tolaasii pathogens. Additionally, the antibacterial effect of this phage cocktail persisted in the second cycle of mushroom growth on the cultivation bed.

• 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.

• KSBB Journal
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• v.7 no.4
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• pp.296-301
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• 1992

A Pseudomonas fluorescens was selected from mushrooms and studied in both batch and fed-batch cultures in order to get maximal biomass concentration. P. fluorescens is an aerobic bacterium and antagonistic to Pseudomonas tolaasii which causes blotch disease on the mushroom cap. P fluarescens and P. tolaasii were identified by Gram staining, gelatin liquefaction, oxidase test, etc. and were characterized by pigment production, temperature sensitivity, salt tolerance and rapid pitting test, etc., Celts of P. fluorescens well in medium containing 30g/L of glucose, whereas the growth was inhibited at the glucose levels at higher than 30g/L. The highest values of specific growth rate and productivity were obtained when using 10g/1 of yeast extract. Optimum concentrations of $NH_4Cl$ and ${(NH_4Cl)}_2SO_4$ for culture were found to be 1.0g/L and 0.1g/L respectively. Optimum concentration of $MgSO_4{\cdot}7H_2O$ used as a sulfursource was 1.0g/L. It was also found that the cell concentrations reached the maximum level when grown on the medium containing 1.0g/L of $KH_2PO_4$ and 0.1g/L of $CaCl_2$. Also, the optimum culture conditions were $30^{\circ}C$ and pH 6.0. Cultivation of P. fluarescens at high dissolved oxygen (DO) concentration led to a decrease of bacterial productivity in batch culture. Maximum productivity was achieved at 40% DO concentration.

• Journal of Mushroom
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• v.12 no.1
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• pp.35-40
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• 2014

Several bacteria are known as the causal agents of diseases of the cultivated button mushroom(Agaricus bisporus) and oyster mushroom(Pleurotus ostreatus). Pseudomonas tolaasii is the causal agent of brown blotch disease of commercial mushrooms. Pseudomonas sp. HC1 is a potent biological control agent to control brown blotch disease caused by Pseudomonas tolaasii. This can markedly reduce the level of extracellular toxins (i.e., tolaasins) produced by Pseudomonas tolaasii, the most destructive pathogen of cultivated mushrooms. To define the optimum conditions for the mass production of the Pseudomonas sp. HC1, we have investigated optimum culture conditions and effects of various nutrient source on the bacterial growth. The optimum initial pH and temperature were determined as pH 5.0 and $20^{\circ}C$, respectively. The optimal culture medium for the growth of tolaasin inhibitor bacterium was determined as follows: 0.9% dextrin, 1.5% yest extract, 0.5% $(NH_4)_2HPO_4$, 4mM $FeCl_3$, and 3.0% cysteine.

• Journal of Mushroom
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• v.13 no.2
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• pp.97-102
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• 2015

This study was conducted to investigate optimum conditions for mass production of antagonistic microbes Pseudomonas azotoformans HC5. P. azotoformans HC5 is a potent biological control agent to control brown blotch disease caused by Pseudomonas tolaasii. This markedly showed the antagonistic activity against P. tolaasii, the most destructive pathogen of cultivated mushrooms. To define the optimum conditions for the mass production of the P. azotoformans HC5, we have investigated optimum culture conditions and effects of various nutrient source on the bacterial growth. The optimum initial pH and temperature were determined as pH 6.0 and $15^{\circ}C$, respectively. The optimal concentration of medium elements for the growth of pathogen inhibitor bacterium was determined as follows: 0.6% adonitol, 1.5% yeast extract, 0.8% $NH_4H_2PO_4$, 5mM $MgSO_4$, and 0.2% asparagine.

• Mycobiology
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• v.28 no.3
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• pp.123-126
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• 2000

Sodium hypochlorite alkaline was tested against Pseudomonas tolaasii causing bacterial blotch on cultivated oyster mushroom (Pleurotus ostreatus). The minimum inhibitory concentration of sodium hypochlorite against P. tolaasii contained active chlorine (AC) at 1.4 mg/l on plate assay. The highest cultivation yield was obtained from the treatment of AC 5.7 mg/l. Treatment of sodium hypochlorite at the rate of higher than AC 11.4 mg/l resulted in reduced yields at the harvest. However, the population of total bacteria on the bed surface treated with AC 5.7 mg/l of sodium hypochlorite was maintained to some extent. Inhibitory concentration against total bacteria on the bed surface was over AC 22.8 mg/l. Mushroom mycelium was damaged and its growth strongly inhibited at the concentration of AC 200 mg/l. Mushroom caps showed yellowish symptom by chemical injury by treatments of AC 74.1 mg/l or higher. Sporocarps infected by P. tolaasii were irrevocable at any concentration of sodium hypochlorite. Routine watering with AC 5.7 mg/l from mushroom initiation to the end of picking resulted in reduced bacterial blotch incidence of 40% and 86% at two mushroom farms. The treatment resulted in higher quality mushroom production compared to that conventionally watered with tap water alone.

• Journal of Microbiology and Biotechnology
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• v.28 no.12
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• pp.2064-2070
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• 2018

Pseudomonas tolaasii 6264 is a representative strain that causes bacterial blotch disease on the cultivated oyster mushroom, Pleurotus ostreatus. Bacteriophages are able to sterilize the pathogenic P. tolaasii strains, and therefore, they can be applied in creating disease-free mushroom cultivation farms, through a method known as "phage therapy". For successful phage therapy, the characterization of phage-resistant strains is necessary, since they are frequently induced from the original pathogenic bacteria in the presence of phages. When 10 different phages were incubated with P. tolaasii 6264, their corresponding phage-resistant strains were obtained. In this study, changes in pathogenic, genetic, and biochemical characteristics as well as the acquired phage resistance of these strains were investigated. In the phylogenetic analyses, all phage-resistant strains were identical to the original parent strain based on the sequence comparison of 16S rRNA genes. When various phage-resistant strains were examined by three different methods, pitting test, white line test, and hemolytic activity, they were divided into three groups: strains showing all positive results in three tests, two positive in the first two tests, and all negative. Nevertheless, all phage-resistant strains showed that their pathogenic activities were reduced or completely lost.

• KSBB Journal
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• v.7 no.2
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• pp.149-153
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• 1992

Pseudomanas fluorescens was selected from mushroom and studied in both batch and continuous culture in order to find out optimum conditions for cultivation. P. fluorescens is an aerobic bacterium and antagonistic to Pseudomonas tolaasii which causes blotch disease on the mushroom cap. Cells of P. fluorescens were grown well on medium containing 30g/L of glucose, whereas the growth was inhibited with the glucose concentration at higher than 30g/L. The highest value of specific growth rate and productivity were obtained when using 10g/L of yeast extract. Optimum concentrations of $NH_4Cl$ and $(NH_4)_2SO_4$ for culture were found to be 1.0g/L and 0.1g/L respectively. Optimum concentration of $MgSO_4{\cdot}7H_2O$ used as a sulfur source was 1.0g/L. It was also found that the cell concentrations were at the maximum level when grown on the medium containing 1.0g/L of $KH_2PO_4$ and 0.1g/L of $CaCl_2$. Also, the optimum culture conditions were $30^{\circ}C$ and pH 6.0. Cultivation of P.fluorescens at high initial dissolved oxygen (D.O) value led to a decrease of bacterial productivity in batch culture. Maximum productivity was achieved at 68 for the initial D.O value.