• 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.

• KSBB Journal
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• v.14 no.2
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• pp.146-152
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• 1999

Screening experiments were carried out in order to select bacteria causing brown blotch disease on the mushroom, Pleurotus ostreatus. Four bacteria causing brown blotch disease were isolated from Pleurotus ostreatus and soils around the mushroom farm. Three strains showing antagonism against brown blotch causing bacteria, A-11, A-20 and A-29 were also isolated through methods pitting test, cross checking and biochemical test, and identified as Pseudomonas fluorescence for A-11 and A-20, and Pseudomonas sp. for A-29, respectively. Colonial morphology test also showed that A-11 and A-29 were appeared as transparent gel with green color, whereas the colony of A-20 showed opaque gel with light green color.

• 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.

• 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.

• 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.

• 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 Society of Plant Pathology Conference
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• 1999.04a
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• pp.46.1-46
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• 1999

• 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.

• The Korean Journal of Mycology
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• v.19 no.3
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• pp.226-230
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• 1991

Since it was firstly identified in Suweon during the growing season in 1986, this fungus of peanut web blotch, Phoma arachidicola, has been widely distributed to the major peanut growing areas throughout the whole country. Disease symptoms appeared on the adaxial surface of peanut like ink drop lesion with net form, and finally turned to dark brown blotch. The fungus isolated was revealed to form dark brown colonies on PD agar at $28^{\circ}C$, resulting 4.5 mm mycelial extension per day. Hyaline conidia were observed to be either aseptate or single septeted, and sized with the ellipsoidal to cylindrical shape $3.0{\times}14.5\;{\mu}m$. The optimum temperature was ranged 24 to $26^{\circ}$ in the grwoth. Virginia type peanut was more resistant to the fungus than Spainish type in the field reaction.

• 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.