• Research in Plant Disease
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• v.9 no.2
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• pp.94-98
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• 2003

To produce antibodies against Pseudomonas tolaasii and P. agarici, lyophilized P. tolaasii and P. agarici ($5{\times}10^7$ cfu/ml) and Freund, s adjuvant were immunized into rabbits 4 times. The specificity and sensitivity of the antibodies were evaluated by immunodiffusion test and indirect enzyme-linked immunosorbent assay (id ELISA). The ${\alpha}$-P. tolaasii antibody was very specific only against P. tolaasii, while ${\alpha}$-P. agarici antibody was not specific and showed a high cross reactivity toward P. tolaasii with detection limit concentration of $2{\times}10^3$ cfu/ml. However, the cross reactivities of ${\alpha}$-P. agarici antibody toward the related species including P. reactans were very low. Our results showed that ${\alpha}$-P. tolaasii and ${\alpha}$-P. agarici antibodies against P. tolaasii and P. agarici, respectively, might be useful for rapid and simple detection of the causal agents of bacterial brown and yellow blotches in cultivated oyster mushrooms.

• The Korean Journal of Mycology
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• v.27 no.2 s.89
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• pp.132-137
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• 1999

This study was carried out to develop the molecular marker for the detection of Pseudomonas tolaasii, a causative agent of bacterial brown blotch disease of oyster mushroom (Pleurotus ostreatus). When several primers designed from repetitive sequences and pectin lyase genes of bacteria were used to produce DNA polymorphism from different Pseudomonas spp. isolated from edible mushrooms, PEU1 primer derived from pectin lyase gene produced polymorphic bands differentiating P. tolaasii strains from other Pseudomonas species. Two bands, 1.0kb and 0.4kb, found commonly in 6 isolates of P. tolaasii were cloned into pGEM-T vector which were designated as pPTOP1 and pPTOP2, respectively, to use as probe. The 0.4 kb insert of pPTOP2 hybridized to only 6 isolates of P. tolaasii, but did not to the other Pseudomonas species. As few as $1.5{\times}10^3$ colony forming unit (cfu) of P. tolaasii could be detected by dot blot hybridization with the cloned 0.4kb DNA in pPTOP2.

• The Korean Journal of Mycology
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• v.27 no.2 s.89
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• pp.119-123
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• 1999

Genetic diversity of Korean isolates of Pseudomonas tolaasii and WLRO (White line reacting organism) was assessed using BOX-, REP-, and ERIC-PCR analysis. P. tolaasii showed nearly identical band patterns among isolates, whereas considerable DNA polymorphism was found among isolates of WLRO. On the basis of dendogram, WLRO is characterized as a complex group with high degree of genetic differentiation. Genetic relatedness based on repetitive DNA regions was low between P. tolaasii and WLRO isolates.

• 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.

• Journal of Mushroom
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• v.1 no.1
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• pp.28-33
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• 2003

Pseudomonas tolaasii causing brown blotch disease was detected by PCR from water samples collected from the oyster mushroom cultivation farms to find the contamination level of the pathogen in water. Sixteen water samples (28.1%) contain less than 1,000 cfu, 31 samples (54.4%) contain 1,001-10,000 cfu, 6 samples (10.5%) contain 10,001-100,000 cfu, and 4 samples (7%) contain of bacteria per milliliter. P. tolaasii-specific DNA band was amplified in 3 samples (5.3%) by nested-PCR and in 20 samples (35.1%) by immunocapture (IC)-nested PCR respectively. These results suggest that IC-nested-PCR was much more sensitive than nested-PCR in detection of P. tolaasii and a quite few waters using for oyster mushroom cultivation were contaminated with P. tolaasii.

• 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.

• Korean Journal Plant Pathology
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• v.11 no.4
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• pp.353-360
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• 1995

Tests were performed on 232 bacterial strains (71 strains of Pseudomonas tolaasii and 161 white line reacting organisms, WLRO) isolated from cultivated mushrooms. As results, P. tolaasii was divided into 5 groups on the basis of the phenotypical characteristics for the strains, and group 3 was the major one including 48 (62%) out of the total 71 strains. WLRO were classified into 23 groups, and group 10 was the major group (65 strains, 30% of the total WLRO tested). A white line was well formed at 22$^{\circ}C$ and at 4 mm distance between P. tolaasii and WLRO colonies in their dual culture on Pseudomonas agar F medium within 36-hr incubation, but not formed at $25^{\circ}C$ even for 72-hr incubation. The morphological, cultural and biological properties of P. tolaasii group 3, and the main group of WLRO, group 10, were different only in the light of pathogenicity. Also group 2 of P. tolaasii had the characteristics similar to group 24 of WLRO which was pathogenic to cultivated mushrooms, suggesting the P. tollaasiii and WLRO strains may be the same species although their white line forming ability and pathogenicity were more or less different from one another. Subculture of the strains in P. tolaasii group 1 induced the variation of their pathogenicity, white line forming ability and utilization of sodium benzoate and sodium tartrate, and thus it can be inferred that they were converted to strains having the characteristics of group 3 or WLRO groups.

• 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.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.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.