The white-line-in-agar test is not specific for the two cultivated mushroom associated pseudomonads, Pseudomonas tolaasii and Pseudomonas "reactans"

A sharply defined white line in vitro forms between the pathogenic form of Pseudomonas tolaasii and another Pseudomonas bacterium, referred to as "reactans". This interaction has been considered as highly specific. However, results presented in this study rise doubt about the strict specificity of this interaction, as some other pseudomonads, associated with the cultivated mushroom Agaricus bisporus, also yielded a white line precipitate when they were streaked towards Pseudomonas tolaasii LMG 2342T. Moreover, some Finnish isolates inducing brown blotch symptoms on mushrooms like P. tolaasii(T), produced a typical white precipitate when streaked towards P. "reactans" LMG5329, even though phenotypical and genotypical features exclude these isolates from the species P. tolaasii. We propose that the white-line-in-agar (WLA) test should no longer be considered as an unequivocal diagnostic trait of P. tolaasii.     

WLIP and tolaasin I, lipodepsipeptides from Pseudomonas reactans and Pseudomonas tolaasii, permeabilise model membranes

The activity of the White Line Inducing Principle (WLIP) and tolaasin I, produced by virulent strains of Pseudomonas reactans and Pseudomonas tolaasii, respectively, was comparatively evaluated on lipid membranes. Both lipodepsipeptides were able to induce the release of calcein from large unilamellar vesicles. Their activity was dependent on the toxin concentration and liposome composition and in particular it increased with the sphingomyelin content of the membrane. Studies of dynamic light scattering suggested a detergent-like activity for WLIP at high concentration (> 27 microM). This effect was not detected for tolaasin I at the concentrations tested (< 28 microM). Differences were also observed in lipodepsipeptides secondary structure. In particular, the conformation of the smaller WLIP changed slightly when it passed from the buffer solution to the lipid environment. On the contrary, we observed a valuable increment in the helical content of tolaasin I which was inserted in the membrane core and oriented parallel to the lipid acyl chains.     

Identification of non-pseudomonad bacteria from fruit bodies of wild agaricales fungi that detoxify tolaasin produced by Pseudomonas tolaasii

Bacterial isolates from wild Agaricales fungi detoxified tolaasin, the inducer of brown blotch disease of cultivated mushrooms produced by Pseudomonas tolaasii. Mycetocola tolaasinivorans and Mycetocola lacteus were associated with fruit bodies of wild Pleurotus ostreatus and wild Lepista nuda, respectively. Tolaasin-detoxifying bacteria belonging to other genera were found in various wild mushrooms. An Acinetobacter sp. was isolated from fruit bodies of Tricholoma matsutake, Bacillus pumilus was isolated from Coprinus disseminatus, and Sphingobacterium multivorum was isolated from Clitocybe clavipes. A Pedobacter sp., which seemed not be identifiable as any known bacterial species, was isolated from a Clitocybe sp. Tolaasin-detoxifying bacteria identified thus far were attached to the surface of mycelia rather than residing within the fungal cells. M. tolaasinivorans, M. lacteus, B. pumilus, the Pedobacter sp., and S. multivorum efficiently detoxified tolaasin and strongly suppressed brown blotch development in cultivated P. ostreatus and Agaricus bisporus in vitro, but the Acinetobacter sp. did so less efficiently. These bacteria may be useful for the elucidation of mechanisms involved in tolaasin-detoxification, and may become biological control agents of mushroom disease.     

Effects of bacteria isolated from a saprophagous rhabditid nematode Caenorhabditis elegans on the mycelial growth of Agaricus bisporus

P. S. GREWAL AND P. HAND. 1992. The effects of 10 species of bacteria isolated from a saprophagous rhabditid nematode Caenorhabditis elegans on mycelial growth of the cultivated mushroom Agaricus bisporus were studied in agar cultures. Bacterial species showed differential effects on the mycelial growth of A. bisporus and the effects also depended upon the mushroom strain (C43, C54 and U3). Bacillus cereus, Bacillus sp. and Enterobacter amnigenus caused significant inhibition in mycelial growth of all three strains of A. bisporus. Pseudomonas aeruginosa, Ps. fluorescens biovar reactans and Ps. maltophilia resulted in a significant increase in mycelial growth of C54 strain. Enterobacter cloacae caused a mean inhibition of about 83% in the linear mycelial extension of the most commonly cultivated mushroom strain U3. Bacillus cereus, Ent. amnigenus and Ent. cloacae produced volatile inhibitory substance(s). This is believed to be the first report about the inhibitory effects of specific bacteria isolated from a saprophagous nematode on the mycelial growth of A. bisporus.     

Pseudomonas tolaasii and tolaasin: comparison of symptom induction on a wide range of Agaricus bisporus strains

Abstract A wide range of Agaricus bisporus , including commercial, wild and hybrid strains, were tested for resistance to brown blotch disease caused by Pseudomonas tolaasii . Effects of toxin and living bacteria were compared. Wild and hybrid A. bisporus ranged in the same order from very poorly to highly susceptible whatever the inoculum type used, tolaasin or bacteria. Symptom aspects induced by both inocula were visually identical, but some differences occurred in response intensity. The data suggest that toxin is probably not the only factor involved in symptom development.     

WLIP and tolaasin I, lipodepsipeptides from Pseudomonas reactans and Pseudomonas tolaasii, permeabilise model membranes

The activity of the White Line Inducing Principle (WLIP) and tolaasin I, produced by virulent strains of Pseudomonas reactans and Pseudomonas tolaasii, respectively, was comparatively evaluated on lipid membranes. Both lipodepsipeptides were able to induce the release of calcein from large unilamellar vesicles. Their activity was dependent on the toxin concentration and liposome composition and in particular it increased with the sphingomyelin content of the membrane. Studies of dynamic light scattering suggested a detergent-like activity for WLIP at high concentration (> 27 μM). This effect was not detected for tolaasin I at the concentrations tested (< 28 μM). Differences were also observed in lipodepsipeptides secondary structure. In particular, the conformation of the smaller WLIP changed slightly when it passed from the buffer solution to the lipid environment. On the contrary, we observed a valuable increment in the helical content of tolaasin I which was inserted in the membrane core and oriented parallel to the lipid acyl chains.     

Genetic characterization of Pseudomonas 'NZI7'--a novel pathogen that results in a brown blotch disease of Agaricus bisporus

AIMS: To characterize a novel pseudomonad isolate capable of causing brown blotch disease of Agaricus bisporus. METHODS AND RESULTS: Using the white-line-in-agar (WLA) assay, fluorescent pseudomonads isolated from a New Zealand mushroom farm were screened for the lipodepsipeptide tolaasin, a characteristic marker of Pseudomonas tolaasii. One isolate, NZI7, produced a positive WLA assay and caused brown lesions of A. bisporus comparable with those produced by Ps. tolaasii. However, genetic analysis suggested that Ps. tolaasii and NZI7 were genetically dissimilar, and that NZI7 is closely related to Pseudomonas syringae. Nucleotide sequence analyses of a gene involved in tolaasin production indicated that similar genes are present in both NZI7 and Ps. tolaasii. CONCLUSION: NZI7 represents a novel Pseudomonas species capable of causing brown blotch disease of A. bisporus. SIGNIFICANCE AND IMPACT OF THE STUDY: Phenotypic identification of Ps. tolaasii based on A. bisporus browning and positive WLA may have limited specificity.     

Left handed alpha-helix formation by a bacterial peptide

The alpha-helix is a common element of secondary structure in proteins and peptides. In eukaryotic organisms, which exclusively incorporate L-amino acids into such molecules, stereochemical interactions make such alpha-helices, invariably right-handed. Pseudomonas tolaasii Paine is the causal organism of the economically significant brown blotch disease of the cultivated mushroom Agaricus bisporus (Lange) Imbach. P. Tolaasii proceduces an extracellular lipodepsipeptide toxin, tolaasin, which causes the brown pitted lesions on the mushroom cap. Circular dichroism studies on tolaasin in a membrane-like environment indicate the presence of a left-handed alpha-helix, probably formed by a sequence of 7 D-amino acids in the peptide. P. tolaasii represents the first reported example of an organism which has evolved the ability to biosynthesize a left-handed alpha-helix.     

Evidence for genotypic differences between the two siderovars of Pseudomonas tolaasii,cause of brown blotch disease of the cultivated mushroom Agaricus bisporus

Sixteen representative isolates of Pseudomonas tolaasii, the causal agent of brown blotch of the cultivated mushroom Agaricus bisporus, were previously assigned to two siderovars (sv1 and sv2) on the basis of pyoverdines synthesized. Each isolate was pathogenic and produced a typical white line precipitate when cultured adjacent to Pseudomonas "reactans" strain LMG 5329. These 16 isolates of P. tolaasii, representing sv1 and sv2, were further characterized using genotypic methods to examine the relationships between the isolates. Rep-PCR studies revealed two distinct patterns from these isolates, which were consistent with the siderovar grouping. Ribotyping differentiated P. tolaasii LMG 2342T (sv1) and PS 3a (sv2) into two distinct ribotypes. A pair of primers, targeted to a 2.1-kb fragment of tl1 (encoding a tolaasin peptide synthetase), yielded the same PCR product from P. tolaasii LMG 2342T (sv1) and PS 22.2 (sv1), but not from PS 3a (sv2). Southern blot analysis indicated that homologues of tl1 are present in PS 3a, but the pattern of hybridization differed from PS 22.2 and LMG 2342T. Sequence determination and analysis of the internally transcribed spacer region ITSI for P. tolaasii LMG 2342T, LMG 6641, and PS 3a strains further supported the presence of the two siderovars. It is concluded that considerable genotypic differences exist among Finnish isolates of P. tolaasii causing brown blotch disease on the cultivated mushroom, which is in agreement with the phenotypic diversity highlighted through previous siderotyping studies.     

In silico study of the ion channel formed by tolaasin I produced by Pseudomonas tolaasii

A toxin produced by Pseudomonas tolaasii, tolaasin, causes brown blotch disease in mushrooms. Tolaasin forms pores on the cellular membrane and destroys cell structure. Inhibiting the ability of tolaasin to form ion channels may be an effective method to protect against attack by tolaasin. However, it is first necessary to elucidate the three-dimensional structure of the ion channels formed by tolaasin. In this study, the structure of the tolaasin ion channel was determined in silico based on data obtained from nuclear magnetic resonance experiments.     

Double-stranded ribonucleic acid in Agaricus bisporus.

Double-stranded ribonucleic acid present in virus-infected mushrooms of Agaricus bisporus has been resolved through polyacrylamide gel electrophoresis into six molecular-weight forms. Identification of these six double-stranded ribonucleic acids in mushrooms by this procedure has proven to be a useful and diagnostic method for viral infection in the cultivated mushroom.     

Identification and characterization of Nip, necrosis-inducing virulence protein of Erwinia carotovora subsp. carotovora

Erwinia carotovora subsp. carotovora is a gram-negative bacterium that causes soft rot disease of many cultivated crops. When a collection of E. carotovora subsp. carotovora isolates was analyzed on a Southern blot using the harpin-encoding gene hrpN as probe, several harpinless isolates were found. Regulation of virulence determinants in one of these, strain SCC3193, has been characterized extensively. It is fully virulent on potato and in Arabidopsis thaliana. An RpoS (SigmaS) mutant of SCC3193, producing elevated levels of secreted proteins, was found to cause lesions resembling the hypersensitive response when infiltrated into tobacco leaf tissue. This phenotype was evident only when bacterial cells had been cultivated on solid minimal medium at low pH and temperature. The protein causing'the cell death was purified and sequenced, and the corresponding gene was cloned. The deduced sequence of the necrosis-inducing protein (Nip) showed homology to necrosis- and ethylene-inducing elicitors of fungi and oomycetes. A mutant strain of E. carotovora subsp. carotovora lacking the nip gene showed reduced virulence in potato tuber assay but was unaffected in virulence in potato stem or on other tested host plants.     

Identification of compounds exhibiting inhibitory activity toward the Pseudomonas tolaasii toxin tolaasin I using in silico docking calculations,NMR binding assays,and in vitro hemolytic activity assays

Using in silico docking calculations, NMR analysis of target–ligand binding, and hemolytic activity assays, we searched a 30,000-compound library for an effective inhibitor of tolaasin I, a Pseudomonas tolaasii toxin that causes virulent infection in mushrooms. Of more than 30,000 compounds screened in silico, two compounds were selected. One of these compounds, sorbitololeic acid, bound to tolaasin I and inhibited its hemolytic activity in vitro. Therefore, sorbitololeic acid can be a potential inhibitor of tolaasin I.     

Application of siderotyping for characterization of Pseudomonas tolaasii and "Pseudomonas reactans" isolates associated with brown blotch disease of cultivated mushrooms

Pyoverdine isoelectric focusing analysis and pyoverdine-mediated iron uptake were used as siderotyping methods to analyze a collection of 57 northern and central European isolates of P. tolaasii and "P. reactans." The bacteria, isolated from cultivated Agaricus bisporus or Pleurotus ostreatus mushroom sporophores presenting brown blotch disease symptoms, were identified according to the white line test (W. C. Wong and T. F. Preece, J. Appl. Bacteriol. 47:401-407, 1979) and their pathogenicity towards A. bisporus and were grouped into siderovars according to the type of pyoverdine they produced. Seventeen P. tolaasii isolates were recognized, which divided into two siderovars, with the first one containing reference strains and isolates of various geographical origins while the second one contained Finnish isolates exclusively. The 40 "P. reactans" isolates divided into eight siderovars. Pyoverdine isoelectric focusing profiles and cross-uptake studies demonstrated an identity for some "P. reactans" isolates, with reference strains belonging to the P. fluorescens biovars II, III, or V. Thus, the easy and rapid methods of siderotyping proved to be reliable by supporting and strengthening previous taxonomical data. Moreover, two potentially novel pyoverdines characterizing one P. tolaasii siderovar and one "P. reactans" siderovar were found.     

Purification of a pore-forming peptide toxin, tolaasin, produced by Pseudomonas tolaasii 6264

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.     

Degradation of acyl-homoserine lactone molecules by Acinetobacter sp. strain C1010

A bacterium C1010, isolated from the rhizospheres of cucumbers in fields in Korea, degraded the microbial quorum-sensing molecules, hexanoyl homoserine lactone (HHSL), and octadecanoyl homoserine lactone (OHSL). Morphological characteristics and 16S rRNA sequence analysis identified C1010 as Acinetobacter sp. strain C1010. This strain was able to degrade the acyl-homoserine lactones (AHLs) produced by the biocontrol bacterium, Pseudomonas chlororaphis O6, and a phytopathogenic bacterium, Burkholderia glumae. Co-cultivation studies showed that the inactivation of AHLs by C1010 inhibited production of phenazines by P. chlororaphis O6. In virulence tests, the C1010 strain attenuated soft rot symptom caused by Erwinia carotovora ssp. carotovora. We suggest Acinetobacter sp. strain C1010 could be a useful bacterium to manipulate biological functions that are regulated by AHLs in various Gram-negative bacteria.     

Erwinia carotovora Evf antagonizes the elimination of bacteria in the gut of Drosophila larvae

Erwinia Virulence Factor (Evf) has been identified in Erwinia carotovora carotovora 15 (Ecc15) as a virulence factor that promotes colonization of the Drosophila larval gut and provokes the triggering of a systemic immune response. Here we have analysed how Evf promotes persistence and colonization of bacteria inside the larval gut. Erwinia evf mutants do not persist in immune-deficient Drosophila, indicating that Evf does not act by counteracting immunity. The results indicated that Evf is not a toxin because various gram-negative bacteria expressing evf can persist without affecting viability of Drosophila larvae. Evf did not appear to be a factor antagonizing a host-specific reaction because in vitro assays failed to reveal detoxifying enzymatic activities against various compounds thought to contribute to the hostile environment of the gut. These findings were corroborated by the observation that Evf is not required for survival in midgut organ cultures. By contrast, bacteria expressing evf allow persistence in trans of bacteria lacking evf indicating that Evf promotes the accumulation of gram-negative bacteria in the anterior midgut by affecting gut physiology.