• 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.

• Korean Journal Plant Pathology
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• v.10 no.3
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• pp.197-210
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• 1994

This experiment was carried out to study the cause of degeneration and rot of cultivated mushroom. Among 597 bacterial isolates derived from the rots of Button mushroom (Agaricus bisporus), Oyster mushroom (Pleurotus ostreatus) and Oak mushroom (Lentinus edodes) collected from markets of 5 cities (Seoul, Suwon, Taegu, Pohang and Pusan) in Korea (1991~1993), 111 bacterial isolates (18.5%) were proved as pathogenic bacteria. These pathogenic bacteria causing bacterial rots of cultivated mushrooms were identified as Pseudomonas tolasii, P. agarici, and Eriwinia sp., and the main causal bacteria were P. tolaasii. P. fluorescens and Klebsiella plenticola were confirmed as saprophytic non-pathogenic bacteria. One hundred fifty nine isolates (Group No. 39) of the 486 saprophytic bacterial isolates were classified as P. fluorescens, and this species was most often found rot area of cultivated mushrooms. P. tolaasii, the causal organism of bacterial blotch, was classified into two groups; One group can be differentiated from the other by the formation of white precipitation band by white line reacting organisms of Pseudomonas Agar F media. P. tolaasii attacked the cultivated mushrooms relatively well at lower incubation temperature such as 5$^{\circ}C$, but P. agarici rarely attack at below 1$0^{\circ}C$. The temperature for the infection commercial cultivated mushrooms by P. agarici was higher than that of P. tolaasii. Optimum temperature for the infection of mushrooms by P. tolaasii and P. agarici were 2$0^{\circ}C$ and $25^{\circ}C$, 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 Korean Journal of Mycology
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• v.26 no.1 s.84
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• pp.60-68
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• 1998

The effect of P. agarici and P. tolaasii causing the bacterial disease of mushrooms on the mycelial growth and fruitbody formation of F. velutipes was evaluated in laboratory. When the pathogenic bacteria was inoculated simultaneously with F. velutipes or 5 days after inoculation of F. velutipes, they significantly deterred both mycelial growth and fruitbody formation of F. velutipes in sawdust culture and showed strong inhibition under high population density. They appeared to be tender or milky in exhibiting symptom on F. velutipes by inoculating the concentration of $10^2{\sim}10^6$ of unit/g media, and their growth seemed to be stopped under $10^8\;cfu/g$ media. On $10^2\;cfu/g$ media of P. agarici and $10^4\;cfu/g$ media of P. tolaasii, there was no effect on the fruitbody yield of F. velutipes. P. tolaasii was more suppressive in the mycelial growth of F. velutipes than P. agarici, while on fruitbodies formation of F. velutipes, P. agarici showed slightly higher inhibition than that of P. tolaasii. When the bacteria was inoculated 10 days after inoculation of F. velutipes, both mycelial growth and fruit body formation were not affected nearly.

• 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.

• 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.

• 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.

• 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 Applied Biological Chemistry
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• v.58 no.4
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• pp.349-354
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• 2015

Brown blotch disease in cultivated mushrooms is caused by Pseudomonas tolaasii, which secretes a lipodepsipeptide, tolaasin. Tolaasin is a pore-forming toxin in the cell membranes, thus destroying the fruiting body structure of mushroom. In this study, we isolated pathogenic bacteria from mushrooms that had symptoms of brown blotch disease. In order to identify these bacteria, their 16S rRNA genes were sequenced and analyzed. Pathogenic bacteria identified as Pseudomonas species were thirty five and classified into five subgroups: P1 to P5. Each subgroup showed different metabolic profile measured by API 20NE kit. Fifty percent of the bacteria were identified as P. tolaasii (P1 subgroup). All five subgroups caused the formation of brown blotches on mushroom tissues and the optimum temperature was 25oC, indicating that they may be able to secrete causal factors, such as tolaasin and similar peptide toxins. These results show that there are at least five different pathogenic Pseudomonas species as blotch-causing bacteria and, therefore, strains from the P2 to P5 subgroups should be also considered and studied as pathogens in order to improve the quality and yield of mushroom production.

• 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.