• Journal of Mushroom
• /
• v.22 no.2
• /
• pp.37-47
• /
• 2024

Fluorescent bacteria were isolated from sporocarps that browned into various mushrooms during survey at places of the production in Korea. We examined the pathogenicity, biodiversity, and genetic characteristics of the 19 strains identified as Pseudomonas tolaasii by sequence analysis of 16S rRNA and White Line Assay. The results emphasize the importance of rpoB gene system, fatty acid profiles, specific and sensitive PCR assays, and lipopeptide detection for the identification of P. tolaasii. As a result of these various analyses, 17 strains (CHM03~CHM19) were identified as P. tolaasii. The phylogenetic analysis based on the 16S rRNA gene showed that all strains were clustered closest to P. tolaasii lineage, two strains (CHM01, CHM02) were not identified as P. tolaasii and have completely different genetic characteristics as a result of fatty acids profile, specific and sensitive PCR, lipopetide detection, rpoB sequence and REP-PCR analysis. Pathogenicity tests showed 17 strains produce severe brown discolouration symptoms to button mushrooms and watersoaking of sporophore tissue within three days after inoculation. But two strains did not produce discolouration symptoms. Therefore, these two strains will be further investigated for correct species identification by different biological and molecular characteristics.

• Research in Plant Disease
• /
• v.28 no.2
• /
• pp.69-81
• /
• 2022

This study was performed to screen the efficacy of antagonistic bacterial isolates from various sources against the bacterial fruit blotch (BFB) causing pathogen (Acidovorax citrulli) in cucurbit crops. In addition, plant growth promoting traits of these antagonistic bacterial isolates were characterized. Two thousand seven hundred ninety-four microorganisms were isolated from the collected samples. Molecular identification revealed two A. citrulli out of 2,794 isolates. In vitro antagonistic results showed that, among the 28 antagonistic bacterial isolates, 24 and 14 bacterial isolates exhibited antagonism against HPP-3-3B and HPP-9-4B, respectively. Antagonistic and growth promotion characterization of the antagonistic bacterial isolates were further studied. Results suggested that, 4 antagonistic bacteria commonly showed both antagonism and growth promotion phenotypes. Moreover, 3 isolates possessed growth promoting activities. Overall results from this study suggests that BFB causing bacterial pathogen (A. citrulli) was suppressed in in vitro antagonism assay by antagonistic bacterial isolates. Furthermore, these antagonistic bacterial isolates possessed growth promotion and antagonistic enzyme production ability. Therefore, data from this study can provide useful basic data for the in vivo experiments which ultimately helps to develop the eco-friendly agricultural materials to control fruit rot disease in cucurbit crops in near future.

• Journal of Applied Biological Chemistry
• /
• v.52 no.1
• /
• pp.28-32
• /
• 2009

The bacterial toxin, tolaasin, causes brown blotch disease on the cultivated mushrooms by collapsing fungal and fruiting body structure of mushroom. Cytotoxicity of tolaasin was evaluated by measuring hemolytic activity because tolaasins form membrane pores on the red blood cells and destroy cell structure. While we investigated the inhibitions of hemolytic activity of tolaasin by $Zn^{2+}$ and $Cd^{2+}$, we found that $Ni^{2+}$ is another antagonist to block the toxicity of tolaasin. $Ni^{2+}$ inhibited the tolaasin-induced hemolysis in a dose-dependent manner and its Ki value was $\sim10$ mM, implying that the inhibitory effect of $Ni^{2+}$ is stronger than that of $Cd^{2+}$. The hemolytic activity was completely inhibited by $Ni^{2+}$ at the concentration higher than 50 mM. The effect of $Ni^{2+}$ was reversible since it was removed by the addition of EDTA. When the tolaasin-induced hemolysis was suppressed by the addition of 20 mM $Ni^{2+}$, the subsequent addition of EDIA immediately initiated the hemolysis. Although the mechanism of $Ni^{2+}$ -induced inhibition on tolaasin toxicity is not known, $Ni^{2+}$ could inhibit any of fallowing processes of tolaasin action, membrane binding, molecular multimerization, pore formation, and massive ion transport through the membrane pore. Our results indicate that $Ni^{2+}$ inhibits the pore activity of tolaasin, the last step of the toxic process.

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

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

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

• The Plant Pathology Journal
• /
• v.37 no.6
• /
• pp.555-565
• /
• 2021

Bacteriophages infecting Acidovorax citrulli, the causal agent of bacterial fruit blotch, have been proven to be effective for the prevention and control of this disease. However, the occurrence of bacteriophage-resistant bacteria is one of hurdles in phage biocontrol and the understanding of phage resistance in this bacterium is an essential step. In this study, we aim to investigate possible phage resistance of A. citrulli and relationship between phage resistance and pathogenicity, and to isolate and characterize the genes involved in these phenomena. A phage-resistant and less-virulent mutant named as AC-17-G1 was isolated among 3,264 A. citrulli Tn5 mutants through serial spot assays and plaque assays followed by pathogenicity test using seed coating method. The mutant has the integrated Tn5 in the middle of a cupin protein gene. This mutant recovered its pathogenicity and phage sensitivity by complementation with corresponding wild-type gene. Site-directed mutation of this gene from wild-type by CRISPR/Cas9 system resulted in the loss of pathogenicity and acquisition of phage resistance. The growth of AC-17-G1 in King's B medium was much less than the wild-type, but the growth turned into normal in the medium supplemented with D-mannose 6-phosphate or D-fructose 6-phosphate indicating the cupin protein functions as a phosphomannos isomerase. Sodium dodecyl sulfa analysis of lipopolysaccharide (LPS) extracted from the mutant was smaller than that from wild-type. All these data suggest that the cupin protein is a phosphomannos isomerase involved in LPS synthesis, and LPS is an important determinant of pathogenicity and phage susceptibility of A. citrulli.

• Journal of Applied Biological Chemistry
• /
• v.60 no.2
• /
• pp.149-153
• /
• 2017

Contamination and growth of Trichoderma, a green mold, on the oak log and wooden chip or sawdust media can severely inhibit the growth of oak mushroom. Chemicals including pesticides and antibiotics are generally not allowed for the control of green mold disease during mushroom cultivation. In this study, bacterial pathogens causing blotch disease on the oyster mushrooms were isolated and their peptide toxins were purified for the control of green mold disease. Strains of Pseudomonas tolaasii secret various peptide toxins, tolaasin and its structural analogues, having antifungal activities. These peptides have shown no effects on the growth of oak mushrooms. When the peptide toxins were applied to the green mold, Trichoderma harzianum H1, they inhibited the growth of green molds. Among the 20 strains of peptide-forming P. tolaasii, strong, moderate, and weak antifungal activities were measured from 8, 5, and 7 strains, respectively. During oak mushroom cultivation, bacterial culture supernatants containing the peptide toxins were sprayed on the aerial mycelia of green molds grown on the surface of sawdust media. The culture supernatants were able to suppress the fungal growth of green molds while no effect was observed on the mushroom growth and production. They changed the color of molds from white aerial mycelium into yellowish dried scab, representing the powerful anti-fungal and sterilization activities of peptide toxins.

• Journal of Mushroom
• /
• v.9 no.4
• /
• pp.135-138
• /
• 2011

'Gonji-2ho' a new variety of oyster mushroom, fitting for the bag culture, was bred by mating between monokaryons isolated from GMPO35338 and Jangpug. In the major characteristics of fruit body, the pilei were thick and dark-gray and the stipes were thick and long with softness. It was great in elasticity and cohesivness of tissue as compared to Suhan-1ho. The optimum temperature for the mycelial growth was around $26{\sim}29^{\circ}C$ and for the pinheading and growth of fruit body was around $14{\sim}18^{\circ}C$. In the bag culture, it was required around 20 days at incubation period and 5 days at primordia formation. The fruit body was grown vital and uniform. The yields were 323.3g/kg bag. This variety has high yielding capacity, cultivation stability and the resistance to the bacterial brown blotch disease.

• The Korean Journal of Mycology
• /
• v.41 no.4
• /
• pp.248-254
• /
• 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.