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Tolaasins are lipodepsipeptides secreted by Pseudomonas tolaasii, the causal agent of brown blotch disease of mushroom, and are the toxins that cause the brown spots. We previously reported that Microbacterium foliorum NBRC 103072T is an effective tolaasin-detoxifying bacterium. In this study, we aimed to characterize the tolaasin-detoxification process of M. foliorum NBRC 103072T. The tolaasin detoxification by M. foliorum NBRC 103072T was carried out by hydrolyzation of tolaasins at two specific sites in the peptide moiety of tolaasins by its cells, and the resulting fragments were released from bacterial cells. The tolaasin-hydrolyzing activity can be extracted by a neutral detergent solution from M. foliorum NBRC 103072T cells. Moreover, tolaasin adsorption to the bacterial cells occurred prior to hydrolyzation of tolaasins, which might contribute to the effective tolaasin detoxification by M. foliorum NBRC 103072T. It is notable that the tolaasin-degradation process by M. foliorum NBRC 103072T is carried out by hydrolyzation at specific sites in the peptide moiety of lipopeptide by bacterial cells as a novel biological degradation process of cyclic lipopeptides. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

From September to December 2018, commercial button mushroom (Agaricus bisporus) farms in central Iran were surveyed to monitor the causal agent(s) of browning and blotch symptoms on mushroom caps. In addition to dozens of pseudomonads (i.e., Pseudomonas tolaasii and Pseudomonas reactans), six slow-growing gram-positive bacterial strains were isolated from blotched mushroom caps. These bacteria presented as creamy white, circular, smooth, nonfluorescent, and shiny colonies with whole margins resembling members of Microbacteriaceae (Actinobacteria). All of the actinobacterial strains were aggressively pathogenic on cut cap surface of two edible mushrooms (i.e., A. bisporus and Pleurotus eryngii), inducing brown pit symptoms 48 h postinoculation. The strains did not induce symptoms on the vegetables tested (i.e., carrot, cucumber, and potato), and they did not affect the growth of mycelium of tested plant-pathogenic fungi (i.e., Acremonium sp., Fusarium spp., and Phytopythium sp.). Phylogeny of 16S ribosomal RNA and multilocus sequence analysis of six housekeeping genes (i.e., atpD, dnaK, gyrB, ppK, recA, and rpoB) revealed that the bacterial strains belong to the actinobacterial genus Mycetocola spp., whereas the species status of the strains remains undetermined. Mushroom-associated Mycetocola species were previously reported to be capable of detoxifying tolaasin, a toxin produced by P. tolaasii, whereas the strains isolated in this study did not show tolaasin detoxification activities. Altogether, this is the first report of a mushroom disease caused by an actinobacterial species, and “bacterial brown pit” was assigned as the common name of the disease.

Tolaasins are lipodepsipeptides secreted by Pseudomonas tolaasii, the causal agent of bacterial blotch on several kinds of cultivated mushrooms. Our previous study reported on tolaasin detoxification by Microbacterium sp. K3-5 as a potential biocontrol of the disease. In this study, the tolaasin-detoxifying activities of various type strains of Microbacterium spp. were evaluated through chemical and biological assays. The bacterial cells of all tested strains of Microbacterium spp. showed tolaasin I-elimination from liquid phase. However, the toxin activities of tolaasins were still retained on the tolaasin-treated bacterial cells of all Microbacterium strains except M. foliorum NBRC 103072T. Furthermore, intact tolaasin I was recovered from the tolaasin-treated bacterial cells of all tested strains except M. foliorum NBRC 103072T. Our data reveal that Microbacterium spp. can be characterized as effective tolaasin I-eliminating bacteria through cell adsorption, but that this adsorption alone is insufficient for actual tolaasin detoxification. The biological degradation process must be needed to carry out the detoxification.

Green mould disease is caused by Trichoderma aggressivum which colonizes mushroom compost and reduces yield. Two Pseudomonas species are associated with mushroom compost: Pseudomonas putida, which stimulates mushroom pinning, and Pseudomonas tolaasii which has a negative effect on crop production. The aim of this work was to characterize T. aggressivum – Pseudomonas interactions as these may be important factors in the development of green mould disease. P. tolaasii supernatant inhibited growth by 57% but P. putida stimulated growth of T.aggressivum by 44%. Tolaasin production was identified in P. tolaasii cultures with a peak at 96 h. Fluorescent microscopy examination of T. aggressivum hyphae revealed that exposure to P. tolaasii supernatant decreased mycelial formation while increasing the abundance of conidia. Label free proteomic analysis of changes in the abundance of T. aggressivum proteins indicated that exposure to P. tolaasii supernatant lead to an oxidative stress response and catabolic enzyme activation (mitochondrial import inner membrane translocase complex (5.7-fold), oxidoreductase (5.2-fold), glucoamylase (5.1-fold)). Exposure of T. aggressivum to P. putida supernatant lead to an increase in the abundance of proteins associated with growth and development (structural constituents of ribosome (20-fold), H/ACA ribonucleoprotein complex subunit (18-fold), DNA binding and nucleosome assembly protein (5.3-fold), and prefoldin (5-fold)). These results indicate that exposure to P. putida can stimulate the growth of T. aggressivum and this interaction may be an important factor in increasing green mould disease in mushroom crops and so reducing yield.

In autumn 2018, cap discoloration and browning symptoms (up to 20% incidence) were observed on commercially grown white button mushrooms (Agaricus bisporus) in two distinct farms located in Shiraz and Marvdasht Counties in Southern Iran. Symptomatic caps (13 and six caps from Shiraz and Marvdasht Counties, respectively) were characterized by visible brown discoloration with no blotch symptoms, bacterial sticky mass and cap wilting. Isolation of bacterial strains from infected cap tissues was performed on yeast-extract peptone glucose agar (YPGA) according to Hamidizade et al. (2020). The resulted bacterial colonies were oyster-white in color, non-fluorescent, domed convex circular with smooth margins 1-2 mm in diameter. A total of six bacterial strains (Shiraz: Ir1002, Ir1003, Ir1004, Ir1005, Ir1007 and Marvdasht: Ir1027) were isolated from distinct mushroom samples. Standard biochemical and phenotypic analyses (Schaad et al. 2001) showed that the bacterial strains were Gram and oxidase negative, catalase positive and facultatively anaerobic, while no capsule or endospore was observed. All strains were positive in urease production, arginine dihydrolase, hydrolysis of tween 80, and utilization of sucrose and D-sorbitol, while they were negative in amylase, cellulose, lecithinase, pectinase, and protease production as well as casein hydrolysis. Based on these phenotypic characteristics, the strains were supposed to be members of Enterobacteriaceae. They also did not induce hypersensitive reaction (HR) on tobacco (Nicotiana tabacum cv. Turkish) leaves nor did they produce tolaasin when streaked side-by-side with "Pseudomonas reactans" strains on King B medium (Osdaghi et al. 2019). Pathogenicity of the strains was evaluated (repeated twice) on fresh caps of white button mushroom using cut-cap method (Hamidizade et al. 2020). Reference strains of Pseudomonas tolaasii (CFBP 8707) and Mycetocola spp. (CFBP 8708) were used as positive controls, while sterile distilled water was used as a negative control. Brown discoloration appeared 24-36 hours post inoculation on cap surfaces while control caps remained asymptomatic. Koch's postulates were accomplished by re-isolation and identification of bacterial strains from the symptomatic caps using colony morphology and Gram staining. For molecular identification, all initial as well as re-isolated strains were subjected to amplification and sequencing of 16S rDNA and gyrB (Yamamoto and Harayama 1995; Hamidizade et al. 2020). Obtained nucleotide sequences were deposited into NCBI GenBank (16S: MZ298620 to MZ298625; gyrB: MZ313184 to MZ313189). BLAST search using the 16S rDNA and gyrB sequences showed that the strains isolated in this study had 97-99% sequence similarity to the reference strains of Cedecea neteri. Phylogenetic analyses also confirmed close relationship of bacterial strains from this study to C. neteri strains. Pure cultures of representative strains Ir1004 (CFBP 8900) and Ir1027 (CFBP 8896) are deposited in CIRM-CFBP culture collection. This is the first report of C. neteri causing brown spot disease on button mushroom in Iran, while the bacterium has previously been reported to cause soft rot on Pholiota nameko (Yan et al. 2018), and yellow sticky disease on Flammulina velutipes (Yan et al. 2019) in China. Further comprehensive investigations will shed a light on the economic impact of the brown spot disease on mushroom industry in Iran.

Pseudomonas tolaasii 6264 is a representative strain that causes bacterial blotch disease on the cultivated oyster mushroom, Pleurotus ostreatus. Bacteriophages are able to sterilize the pathogenic P. tolaasii strains, and therefore, they can be applied in creating disease-free mushroom cultivation farms, through a method known as "phage therapy". For successful phage therapy, the characterization of phage-resistant strains is necessary, since they are frequently induced from the original pathogenic bacteria in the presence of phages. When 10 different phages were incubated with P. tolaasii 6264, their corresponding phage-resistant strains were obtained. In this study, changes in pathogenic, genetic, and biochemical characteristics as well as the acquired phage resistance of these strains were investigated. In the phylogenetic analyses, all phage-resistant strains were identical to the original parent strain based on the sequence comparison of 16S rRNA genes. When various phage-resistant strains were examined by three different methods, pitting test, white line test, and hemolytic activity, they were divided into three groups: strains showing all positive results in three tests, two positive in the first two tests, and all negative. Nevertheless, all phage-resistant strains showed that their pathogenic activities were reduced or completely lost.

Tolaasins are antimicrobial lipodepsipeptides. Here, we report the tolaasins-detoxifying properties of Microbacterium sp. K3-5 (K3-5). The detoxification of tolaasins by K3-5 was performed by hydrolyzation of cyclic structure of tolaasins depending on the tolaasin-K3-5 cell interaction. Our data suggest that the cyclic structure of tolaasins is critical for its interaction to target cells.

Bacterial brown blotch disease is one of the most serious bacterial diseases in cultivated mushrooms. Fluorescent pseudomonas isolated from different lesions on the caps of cultivated Agaricusbisporus were identified as strains of Pseudomonas tolaasii and the poorly described Pseudomonas “reactans”. Anaccurate identification of P. tolaasii was carried out by sequencing 16SrDNA gene. Phenotypically the isolates were heterogeneous, which further hindered their correct identification. Regarding P. “reactans”, this bacterial name is not regarded as a valid name and there is no type strain. In this work we have named as P. reactans type those isolates that induce the precipitation of tolaasin. This would suggest that we are not dealing with one species but a group of bacteria. Both, P. tolaasii and P. reactans were shown to be pathogenic in the assays performed, so the aetiology of the disease can be manifold.The incidence of blotch disease in Spanish mushroom crops was evaluated to affect up 15% of the total yield, with the highest losses detected for the second flush. The development of the disease wasassociated with high relative air humidity registered on the irrigation day closest to the harvesting of the second flush, which may lead to condensation on the caps as soon as the air temperature changesif there is no adequate ventilation. Modifying the environmental conditions by increasing the temperature prior to irrigation and moderating the manoeuvres of the drying processhelped control bacterial blotch disease.

Pseudomonas sp. 11K1, originally isolated from rhizosphere, possesses inhibitory activity against plant pathogenic fungi and bacteria. Herein, the genome of strain 11K1 was sequenced and subjected to in silico, mutational, and functional analyses. The 11K1 genome is 6,704,877 bp in length, and genome mining identified three potential cyclic lipopeptide (CLP) biosynthetic clusters, subsequently named brasmycin, braspeptin, and brasamide. Insertional and deletion mutants displayed impaired brasmycin and braspeptin production, and lost antifungal activity, but retained antibacterial activity against Xanthomonas oryzae. The structures of these two active CLPs were predicted based on adenylation (A) domains. Brasmycin is composed of nine amino acids and belongs to the syringomycin class, while braspeptin is a 22 amino acid cyclic peptide belonging to the tolaasin group. Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) mass spectrometry analysis revealed that brasmycin and braspeptin have different molecular weights compared with known syringomycin and tolaasin members, respectively. Mutation of brasmycin and braspeptin gene clusters affected both biofilm formation and colony morphology. Collectively, these results indicate that Pseudomonas sp. 11K1 produces two novel CLPs that may help bacteria compete for nutrients and niches in the environment.

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.

Symbiosis The white button mushrooms that many of us enjoy eating are subject to brown blotch disease caused by the bacterium Pseudomonas tolaasii . These bacteria are spread by means of biosurfactants, and damage is caused by a family of pore-forming cyclic lipopeptides called tolaasins. However, protective bacteria are also found on button mushrooms, and Hermenau et al. show how helper bacteria of the genus Mycetocola protect their host. Metabolic profiling, mass spectroscopy, structural characterization, and infection assays show that tolaasin is linearized by the helper bacteria. The authors also identify the enzyme responsible and show that a biosurfactant involved in pathogen spreading is also a cyclic lipopeptide that is neutralized by linearization. Understanding these protective mechanisms could allow the development of biocontrol strategies to stop spoilage. Proc. Natl. Acad. Sci. U.S.A. 10.1073/pnas.2006109117 (2020).

Brown blotch disease of mushroom caused by Pseudomonas tolaasii, often results of severe damage to mushroom tissues. In this study, the biocontrol mechanisms mediated by endofungal bacteria Pseudomonas sp. De1, Bacillus sp. De3, Pseudomonas sp. Bi1 and Pantoea sp. Ma3 previously isolated from wild growing mushrooms against Pseudomonas tolaasii Pt18 were demonstrated. Our results provide evidence that all bacterial strains can colonize mycelia of Agaricus bisporus. All endogungal bacteria except Pseudomonas sp. Bi1 contained one or more genes encoding antibiotic production. The non-volatile compounds of endofungal bacteria significantly inhibited the biofilm formation by P. tolaasii Pt18. Results showed that all strains except Pantoea sp. Ma3 are capable of detoxify tolaasin, a major virulence factor produced by P. tolaasii Pt18. We also showed that all endofungal bacteria tested were able to significantly decrease the browning symptoms of pretreated-mushroom caps compared to inoculated caps with P. tolaasii alone. AbPPO3 gene expression levels in mycelia and fruiting bodies of A. bisporus significantly increased after P. tolaasii Pt18 inoculation. Our findings indicated that The relative expression level of AbPPO3 gene decreased in the mushroom mycelia and fruiting bodies pretreated with Pantoea sp. Ma3 and Pseudomonas sp. Bi1 strains, and the levels were significantly lower than mushroom tissues treated with P. tolaasii Pt18 alone. Overall, the findings presented in this study revealed the efficacy of the selected endofungal bacteria in biocontrol activity against P. tolaasii and the possible use of these strains as a safe microbial agents in mushroom brown blotch disease management.

Bacteria in the diverse Pseudomonas fluorescens group include rhizosphere inhabitants known for their antifungal metabolite production and biological control of plant disease, such as Pseudomonas protegens Pf-5, and mushroom pathogens, such as Pseudomonas tolaasii. Here, we report that strain Pf-5 causes brown, sunken lesions on peeled caps of the button mushroom (Agaricus bisporus) that resemble brown blotch symptoms caused by P. tolaasii. Strain Pf-5 produces six known antifungal metabolites under the control of the GacS/GacA signal transduction system. A gacA mutant produces none of these metabolites and did not cause lesions on mushroom caps. Mutants deficient in the biosynthesis of the antifungal metabolites 2,4-diacetylphloroglucinol and pyoluteorin caused less-severe symptoms than wild-type Pf-5 on peeled mushroom caps, whereas mutants deficient in the production of lipopeptide orfamide A caused similar symptoms to wild-type Pf-5. Purified pyoluteorin and 2,4-diacetylphloroglucinol mimicked the symptoms caused by Pf-5. Both compounds were isolated from mushroom tissue inoculated with Pf-5, providing direct evidence for their in situ production by the bacterium. Although the lipopeptide tolaasin is responsible for brown blotch of mushroom caused by P. tolaasii, P. protegens Pf-5 caused brown blotch–like symptoms on peeled mushroom caps through a lipopeptide-independent mechanism involving the production of 2,4-diacetylphloroglucinol and pyoluteorin.

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 , 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% , 4mM , and 3.0% cysteine.

Brown blotch of cultivatable mushrooms is a disease caused by the small peptide toxin (tolaasin) secreted by Pseudomonas tolaasii. Here we found that the wild type tolassin-producing P. tolaasii stain 6264 was capable of infection in Arabidopsis thaliana cotyledons, causing chlorotic symptoms and growth arrest as a result of bacterial proliferation. Seven virulence-attenuated mutants of P. tolaasii were isolated from the Agaricus bisporus screen using 2512 mariner-based transposon insertion mutants, and all of them displayed reduced virulence and bacterial proliferation in Arabidopsis infection as well. The transposon was inserted within the genes for tolassin biosynthesis and amino acid biosynthesis, and within an intergenic region between the genes of unknown function. The finding that some virulence factors are commonly required for both Agaricus and Arabidopsis infections suggests that Arabidopsis could be exploited to study the host-pathogen interaction involving P. tolaasii.

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.

Brown blotch disease of cultivated mushrooms is caused by Pseudomonas tolaasii, which secretes the bacterial toxin, tolaasin. Tolaasin is a peptide toxin that causes pore formation in the plasma membrane of mushroom cells. Forty-two strains of pathogenic bacteria causing brown blotch or similar diseases were isolated from mushrooms showing disease symptoms. To characterize these bacteria, the genes of 16S rRNA were sequenced and analyzed. Thirty-three strains were identified as five different species of Pseudomonas. Of these, 23 were identified as P. tolaasii and named as P1-type pathogens. Because the strains identified as P. tolaasii were major pathogens that cause the brown blotch disease, phylogenetic analyses of these pathogens were conducted by the neighbor-joining method. These strains comprised three phylogenetic subtypes, P1α (6 strains), P1β (16 strains), and P1γ (1 strain). Biological characterizations of the isolated bacteria were performed and confirmed that all three subtypes were able to cause the disease by forming blotches on the surface of the mushroom tissue. However, hemolytic activities were observed in the P1α and P1γ strains, but not in the P1β strains. These results imply that remarkable diversity exists among the various strains of P. tolaasii, each strain showing distinct biological characters.

Pseudomonas sp. strain DF41 produces a lipopeptide, called sclerosin that inhibits the fungal pathogen Sclerotinia sclerotiorum . The aim of the current study was to deduce the chemical structure of this lipopeptide and further characterize its bioactivity. Mass spectrometry analysis determined the structure of sclerosin to be CH3-(CH2)6-CH(OH)-CH2-CO-Dhb-Pro-Ala-Leu/Ile-Ala-Val-Val-Dhb-Thr-Val-Leu/Ile-Dhp-Ala-Ala-Ala-Val-Dhb-Dhb-Ala-Dab-Ser-Val-OH, similar to corpeptins A and B of the tolaasin group, differing by only 3 amino acids in the peptide chain. Subjecting sclerosin to various ring opening procedures revealed no new ions, suggesting that this molecule is linear. As such, sclerosin represents a new member of the tolaasin lipopeptide group. Incubation of S. sclerotinia ascospores and sclerotia in the presence of sclerosin inhibited the germination of both cell types. Sclerosin also exhibited antimicrobial activity against Bacillus species. Conversely, this lipopeptide demonstrated no zoosporicidal activity against the oomycete pathogen Phytophthora infestans . Next, we assessed the effect of DF41 and a lipopeptide-deficient mutant on the growth and development of Caenorhabditis elegans larvae. We discovered that sclerosin did not protect DF41 from ingestion by and degradation in the C. elegans digestive tract. However, another metabolite produced by this bacterium appeared to shorten the life-span of the nematode compared to C. elegans growing on Escherichia coli OP50.

The secondary metabolite mediating the GacS-dependent growth-inhibitory effect exerted by the rice rhizosphere isolate Pseudomonas putida RW10S2 on phytopathogenic Xanthomonas species was identified as white-line-inducing principle (WLIP), a member of the viscosin group of cyclic lipononadepsipeptides. WLIP producers are commonly referred to by the taxonomically invalid name “ Pseudomonas reactans ,” based on their capacity to reveal the presence of a nearby colony of Pseudomonas tolaasii by inducing the formation of a visible precipitate (“white line”) in agar medium between both colonies. This phenomenon is attributed to the interaction of WLIP with a cyclic lipopeptide of a distinct structural group, the fungitoxic tolaasin, and has found application as a diagnostic tool to identify tolaasin-producing bacteria pathogenic to mushrooms. The genes encoding the WLIP nonribosomal peptide synthetases WlpA, WlpB, and WlpC were identified in two separate genomic clusters ( wlpR-wlpA and wlpBC ) with an operon organization similar to that of the viscosin, massetolide, and entolysin biosynthetic systems. Expression of wlpR is dependent on gacS , and the encoded regulator of the LuxR family (WlpR) activates transcription of the biosynthetic genes and the linked export genes, which is not controlled by the RW10S2 quorum-sensing system PmrR/PmrI. In addition to linking the known phenotypes of white line production and hemolytic activity of a WLIP producer with WLIP biosynthesis, additional properties of ecological relevance conferred by WLIP production were identified, namely, antagonism against Xanthomonas and involvement in swarming and biofilm formation.

Pseudomonas tolaasii, the causal organism of brown blotch disease of Agaricus bisporus and of the yellowing of Pleurotus ostreatus, was shown to produce in culture tolaasin I (1), tolaasin II (2), and five other minor metabolites, tolaasins A, B, C, D, and E (3-7). These compounds were demonstrated to be important in the development of the disease symptoms. This paper reports on the structural elucidation, based essentially on NMR studies and MS spectra, and biological activity of the above lipodepsipeptides (3-7). All the above analogues showed differences in the peptide moiety, as observed in other lipodepsipeptides of bacterial origin, and maintained the beta-hydroxyoctanoyl phi chain at the N-terminus, except tolaasin A, in which the acyl moiety was a gamma-carboxybutanoyl phi moiety. Among the target microorganisms used (fungi, yeast, and bacteria) the Gram-positive bacteria were the most sensitive, although the antimicrobial activity appeared to be correlated to the structural modification in the different analogues. The structure-activity relationships of these toxins are discussed.

Aims: The present study describes PCR assays to detect specifically Pseudomonas tolaasii from various samples. Methods and Results: Two sets of PCR primers were developed to amplify genes required for tolaasin production. Only a PCR product of 449 bp or 249 bp was produced in PCR reactions with the Pt-1A/Pt-1D1 or Pt-PM/Pt-QM primer sets, respectively, and DNA and cells of Ps. tolaasii. Nested and immunocapture-nested PCR could detect to 3 cells of Ps. tolaasii and amplify the Ps. tolaasii-specific DNA from a sample containing 10 000 times more other bacterial cells than Ps. tolaasii, respectively. Conclusions: The PCR assays are simple, rapid and reliable methods for detection and identification of Ps. tolaasii. Significance and Impact of the Study: The protocols can effectively distinguish Ps. tolaasii from other bacteria and detect Ps. tolaasii from various samples for studying ecology of the bacterium and preventing the use of contaminated water or spawn or medium in mushroom cultivation.

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.

Summary 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 2342 T (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 2342 T (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 2342 T . Sequence determination and analysis of the internally transcribed spacer region ITS1 for P. tolaasii LMG 2342 T , 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.

Cyclic lipopeptides (CLPs) are synthesized by nonribosomal peptide synthetases (NRPS), which are often flanked by LuxR‐type transcriptional regulators. Pseudomonas sp. CMR12a, an effective biocontrol strain, produces two different classes of CLPs namely sessilins and orfamides. The orfamide biosynthesis gene cluster is flanked up‐ and downstream by LuxR‐type regulatory genes designated ofaR1 and ofaR2, respectively, whereas the sessilin biosynthesis gene cluster has one LuxR‐type regulatory gene which is situated upstream of the cluster and is designated sesR. Our study investigated the role of these three regulators in the biosynthesis of orfamides and sessilins. Phylogenetic analyses positioned OfaR1 and OfaR2 with LuxR regulatory proteins of similar orfamide‐producing Pseudomonas strains and the SesR with that of the tolaasin producer, Pseudomonas tolaasii. LC‐ESI‐MS analyses revealed that sessilins and orfamides are coproduced and that production starts in the late exponential phase. However, sessilins are secreted earlier and in large amounts, while orfamides are predominantly retained in the cell. Deletion mutants in ofaR1 and ofaR2 lost the capacity to produce both orfamides and sessilins, whereas the sesR mutant showed no clear phenotype. Additionally, RT‐PCR analysis showed that in the sessilin cluster, a mutation in either ofaR1 or ofaR2 led to weaker transcripts of the biosynthesis genes, sesABC, and putative transporter genes, macA1B1. In the orfamide cluster, mainly the biosynthesis genes ofaBC were affected, while the first biosynthesis gene ofaA and putative macA2B2 transport genes were still transcribed. A mutation in either ofaR1, ofaR2, or sesR genes did not abolish the transcription of any of the other two.

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. Conclusions: 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.

A Gram-positive bacterium was isolated from a rottingPleurotus ostreatus fruiting body that markedly reduced the level of extracellular toxins (i.e., tolaasins) produced byPseudomonas tolaasii, the most destructive pathogen of cultivated mushrooms. The isolated bacterium is saprophytic but not parasitic nor pathogenic toP. ostreatus. A low ratio, ca. 10−3 cells of the isolated bacterium for oneP. tolaasii cells, was sufficient for detoxification in vitro. Inoculation of the isolated bacterium prevents the development of bacterial disease inP. ostreatus andAgaricus bisporus. The suppression of the disease development, however requires the initial cell density equivalent to ca. 10−1 cells of the isolated bacterium for one cells of the pathogen. The effects is ascribed to the inactivation of tolaasin by the live, suppressive bacterial cells, and not to metabolites secreted from the organism into culture media. Examination by conventional bacteriological tests and with testing kits, i.e., MicroStationTMSystem Release 3.5 (Biolog Inc., Hayward, CA), ATB Expression (bioMerieux Inc. Japan) and VITEK (bioMerieux Inc. Japan), failed to assign the organism to any defined bacterial genus. The suppressive bacterium may be useful in future for the development of biocontrol system and/or the construction of genetically modified edible fungi resistant to the disease caused byP. tolaasii.

Pseudomonas tolaasii strain PT814 produces extracellular toxins, tolaasins, and a volatile toxin, tovsin, that are responsible for the induction of brown blotch and rotting, respectively, in a cultivated mushroom, Pleurotus ostreatus. Insertions of single transposon mini-Tn 5km 1 into the chromosome of P. tolaasii strain PT814 generated mutants that are pleiotropically defective in tolaasin and protease production, and altered in colony morphology. The mutants, however, produce tovsin at the level of wild-type. Variants phenotypically similar to the pleiotropic mutants of P. tolaasiistrain PT814 spontaneously occurred in P. tolaasii strain S8501 at 22–30°C in vitro. The occurrence of variants was significantly reduced in the presence of extracts of P. ostreatus or at a temperature of 15–20°C The rtpA gene (rtpA = regulator gene of tolaasin production and other pleiotropic traits) isolated from a P. tolaasii strain PT814 gene library restored the wild-type phenotype in both the mini-Tn5 km 1 insertion and spontaneous mutants, mini-Tn5 km 1 insertions were also located in the allele of rtpA. Nucleotide sequencing of the rtpA DNA revealed an open reading frame of 2,751 bp predicted to encode a protein consisting of 917 amino acid residues with a molecular mass of 100.6 kDa and displaying the conserved amino acid sequence of both sensor and receiver domains of “bacterial two-component regulators.” The data suggest that the machinery responding to environmental stimuli is essential for the pathogenic interaction of P. tolaasii with the mushroom.

Strains representing the fluorescent plant pathogenic Pseudomonas spp., Ps. agarici, Ps. asplenii, Ps. avellanae, Ps. beteli, Ps. caricapapayae, Ps. cichorii, Ps. corrugata, Ps. ficuserectae, Ps. flectens, Ps. fuscovaginae, Ps. marginalis, Ps. meliae, Ps. savastanoi, Ps. syringae, Ps. tolaasii and Ps. viridiflava were tested for biocidal activity using Aspergillus niger as assay organism. Inhibitory behaviour was found in strains of Ps. asplenii, Ps. blatchfordae, Ps. cichorii, Ps. corrugata, Ps. fuscovaginae, Ps. marginalis, Ps. marginalis pv. pastinacea, Ps. syringae pv. syringae, Ps. syringae pv. aptata, Ps. syringae pv. atrofaciens, Ps. syringae pv. lapsa, Ps. tolaasii, and strains of a Pseudomonas sp. pathogenic to Actinidia, in the Ps. savastanoi genomic sp. Antifungal activity could be identified with the production of members of the syringomycin family of toxins by strains in Ps. syringae, Ps. asplenii and Ps. fuscovaginae. These toxin reactions support suggestions made elsewhere of the synonymy of the latter two species. In a preliminary characterization using tests for stability to heat, protease, acid and alkaline treatments, unknown toxins consistent with syringomycin-like toxins the strains from Actinidia speciesColour RGB 0,0,128. The toxins from Ps. cichorii and from Ps. corrugata differed in their reactions from all other agents. Pseudomonas tolaasii produces the antifungal compound tolaasin. The white line reaction with ‘Ps. reactans’, a test for tolaasin production by strains of Ps. tolaasii, was confirmed as specific for this compound. Some of these low molecular weight toxins may be produced by some of these plant pathogenic strains.

Syringomycin E (SR-E), syringotoxin (ST) and two forms (SP 22 -A and SP 25 -A) of syringopeptin (SP), the main lipodepsipeptides produced by strains of Pseudomonas syringae pv. syringae , were investigated for their biological activities. All toxins caused lysis of sheep erythrocytes, but SPs were more active than SR-E and ST. The lytic concentrations were 0.8 and 0.6 μ M for SP 22 -A and SP 25 -A and 1.4 and 5.9 μ M for SR-E and ST, respectively. The effect of toxins on the electrolyte leakage from carrot tissues and on the viability of potato tuber tissues confirmed the higher phytotoxicity of SPs in comparison to the SR-E or ST. A relevant effect on electrolyte leakage was induced by 0.4 μ M SP 22 -A and 10 μ M ST, respectively. The spectrum of antimicrobial activity indicated that 50 μ M toxin solutions did not inhibit the growth of Gram-negative bacteria. Gram-positive bacteria were sensitive to SPs but in contrast, SR-E and ST had no relevant effect on the growth of these bacteria. In particular, the growth of Bacillus megaterium was inhibited by 1.56 μ M SP 22 -A and 3.12 μ M SP 25 -A, but was unaffected by 50 μ M SR-E or ST. The effects of toxins on fungi were variable. For example, 6.25 μ M SR-E or ST, 12.5 μ M SP 22 -A and 37.5 μ M SP 25 -A inhibited the growth of the yeast Rhodotorula pilimanae . The mycelial growth of the plant pathogenic fungus Botrytis cinerea was inhibited by 1.6 μ M SP 25 -A, 12.5 μ M SP 22 -A, 18.7 μ M SR-E and 25 μ M ST. Furthermore, SR-E was the most active toxin in inhibiting conidial germination and germ tube elongation of this fungus.

A bacterial disease occurred on fruiting bodies ofLentinula edodes that formed outdoors onQuercus bedlogs during winter. The pathogen was identified asPseudomonas tolaasii based on morphological and bacteriological characteristics. Symptoms exhibited by infected fruiting bodies ranged from mild browning to severe necrotic cavities that characteristically developed in the cap tissue along the periphery of the attachment area to the stalk. The mode of symptom development was greatly influenced by the internal tissue structure of fruiting bodies. Multiplication of bacterial cells within the fruiting bodies was strictly intercellular and thus differed from previously reported bacterial disease ofL. edodes incited by an unidentified rod-shaped bacterium. The present strain ofP. tolaasii was capable of attacking theL. edodes mycelium in the inner bark and outer sapwood regions and caused lysis of heavily infected hyphae.

新潟県下の栽培ヒラタケの黄褐変腐敗症を示す病斑部から病原細菌を分離し,Pseudomonas tolaasii Paineと同定した。本細菌はヒラタケのほかエノキタケやマッシュルームなど数種のキノコに病原性を示した。また,培養ろ液は,キノコに対して病斑類似斑を形成し,植物に対しては毒性を示し,微生物の増殖を阻害した。本毒素に高度感受性を示したエノキタケ,ジャガイモ切片,P. syringae pv. coronafaciensは,毒素の簡易検出や活性測定に有用であった。毒素を得る目的で,菌株,培地,温度,培養日数と毒素生産性との関係を調べ,それぞれにおける生産最適条件を明らかにした。また,Adsorbex RP-18カートリッジを用いた簡便な毒素の単離法を確立した。本法によって8種の毒素を単離するとともに,毒素4および6をそれぞれtolaasin IおよびIIと同定した。主毒素である4は,ヒラタケ,エノキタケ,ジャガイモ切片にそれぞれ125, 16,4ppmの濃度で褐変を誘導した。毒素4は植物病原微生物にも抗菌活性を示したが,その程度は微生物の種によって異なった。感受性は一般に細菌より糸状菌で高く,生育阻害最小濃度は前者で16&500ppm,後者で4&16ppmであった。

Pseudomonas tolaasii causes brown blotch disease in cultivated mushrooms by producing tolaasin, a peptide toxin, which forms pores on the membrane and disrupts the cellular and fruiting body structures of mushrooms. For the biological control of this mushroom disease, virulent bacteriophages of P. tolaasii were isolated from the sewage of Cheongju, Korea. Twenty-one phages were isolated from four different locations, and their toxicities to host bacteria were measured by inspecting the turbidity and size of their plaques. They were divided into three categories on the basis of their toxicities to host bacteria. In order to test if these phages can be used for the biological control of mushroom diseases, a pitting test was performed. The surfaces of mushroom caps were inoculated with both pathogenic bacteria and their phages. Phage toxicity was analyzed by measuring the size of the blotches that formed on the surface of mushrooms, because these sizes are representative of the amount of tolaasin peptide produced by pathogenic bacteria in the presence of bacteriophages. The formation of blotches was completely blocked by co-incubated phages. These results show that phages can sterilize pathogenic bacteria in mushroom tissues as well as be useful for the biological control of brown blotch disease. The optimum conditions for the bactericidal activity of the phages were also determined.

The extracellular lipodepsipeptide toxin tolaasin is the primary disease determinant of pathogenicity of Pseudomonas tolaasii on the cultivated mushroom, Agaricus bisporus. Transposon mutagenesis of P. tolaasii NCPPB 1116 with Tn5-generated 5000 chromosomal insertions of which 35 (0.7%) were tolaasin negative and 12 (0.25%) produced a reduced amount of tolaasin. In addition, TnphoA mutagenesis yielded a single tolaasin-negative mutant which was phoA active. Restriction enzyme mapping of mutant DNAs by Southern hybridization analysis revealed that the majority of Tn5 insertions were confined to a single genetic locus of approximately 65 kbp. Pulsed-field gel electrophoresis of representative Tn5 mutant DNAs showed that this region is at one end of a 640 kbp PacI chromosomal fragment and that the P. tolaasii genome is 6.7 Mbp. SDS-PAGE analysis of protein extracts from wild-type P. tolaasii demonstrated the presence of three high-molecular-weight proteins (designated TL1, TL2 and TL3). Alterations in the presence of these proteins, as well as apparently truncated forms of the 465 kDa (TL1), 440 kDa (TL2) and 435 kDa (TL3) proteins were observed in some mutants, enabling the direction and order of the transcriptional units to be determined. Two other Tn5 mutations were also identified which resulted in a tolaasin-negative phenotype, but which did not affect the expression of TL1, TL2, or TL3. One of these mutants is linked to the TL-cluster, but the other is located outside this region. It is concluded that at least five genetic loci, including those encoding TL1, TL2 and TL3, are required for tolaasin synthesis.

Pleurotus ostreatus is one of the most extensively cultivated mushrooms in the world; however, the success of cultivation often depends on the proliferation of different bacterial pathogens. Pseudomonas tolaasii is thought as the major cause of brown blotch disease of Agaricus bisporus and yellowing of Pleurotus ostreatus. In this study we examined the pathogenicity and assessed the industrial damage causing effect of 41 Pseudomonas strains isolated from deformed, yellowing oyster mushroom (P. ostreatus) sporocarps. Identification of the isolates at species level by the partial sequence analysis of the hypervariable region of the rpoB gene revealed nine Pseudomonas sps. We analyzed the presence of the tolaasin gene-cluster, the production of fluorescent pigments, the oxidase- and nitrite reductase activities, the growth at restrictive temperatures and the carbon source utilizing abilities of each strain. Complex lipopeptide production (including tolaasin) was examined with thin layer chromatography and a novel in vitro necrosis-test was developed and evaluated for the investigation of the pathogenic effect of Pseudomonas strains. Our results underline the importance of extracellular enzyme production in the sporocarp decaying process. Strong correlations were found between the secretion of trypsin-like proteases and lipases and the necrotic effect of these bacteria. All the results clearly established that besides Ps. tolaasii, Ps. fluorescens biovar V strains were pathogenic to P. ostreatus and cause serious losses during mushroom production. Our results underline the importance of extracellular enzyme production in the sporocarp decaying process, especially the trypsin-like proteases and lipases.

Tolaasin, 1.9 kDa peptide toxin, is produced by Pseudomonas tolaasii. It forms pores on the membrane and thus destroys cellular structure, causing brown blotch disease on the cultivated mushroom. Previously, we showed that the tolaasin-induced pore formation required the molecular multimerization and the multimerization of tolaasin was blocked by the treatment of various tolaasin-inhibitory factors (TIF). This was successful to prevent from causing brown blotch disease. Various TIF's blocked effectively the tolaasin-induced hemolysis at 1-100 μM. The tolaasin-induced hemolysis was dependent on the temperature and pH; however, TIF always inhibited hemolysis. In the pitting test using mushrooms of Agaricus bisporus, tolaasin treatment caused brown blotches on the surface of mushroom and the addtion of TIF at 0.1-10 mM completely blocked the blotch formation by tolaasin. Since TIF's were selected from various food additives, the effect and mechanism will be different on the tolaasin-induced hemolysis. In this study, we have measured the synergic effect of various TIF's on the inhibition of tolaasin toxicity. When TIF-9 and TIF-11 were added, tolaasin-induced hemolysis was effectively inhibited. In the presence of both TIF's, the inhibition was increased to 2.5 times compared to the inhibition obtained by any one of them. Similar synergic effect was also measured by the additions of TIF-9 and TIF-16. The inhibition was also increased more than 3 times. However, TIF-10 and TIF-11 did not show any synergic effect. These results showed that there are synergic effects among various TIF's and the effect was dependent on the type and concentration of TIF.

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.

The biological properties and spectrum of activity of tolaasin, a lipodepsipeptide toxin produced by the mushroom pathogen Pseudomonas tolaasii, were investigated. An erythrocyte lysis bioassay was developed and used to assay tolaasin activity. Tolaasin-induced haemolysis was dose dependent, maximal between pH 6·0 and 7·0, increased with temperature and was inhibited by the addition of divalent metal ions. Tolaasin production commenced during exponential growth of P. tolaasii and continued into stationary phase. Culture filtrate from wild type P. tolaasii was able to pit mushroom tissue and lyse erythrocytes, whereas culture filtrate from a tolaasin defective mutant had no lytic activity. Ultrastructural studies showed disruption of the Agaricus bisporus plasma membrane and vacuole membranes by both P. tolaasii and tolaasin, but not by a tolaasin defective mutant. Pre-incubation of tolaasin with multilamellar liposomes resulted in loss of lytic activity and osmotic protectants prevented tolaasin-induced haemolysis, demonstrating that tolaasin partitions into membranes, forms pores in erythrocyte membranes and causes lysis by a colloid osmotic mechanism. Tolaasin was phytotoxic when infiltrated into leaves of Nicotiana tabacum and was shown to be active against a range of basidiomycetes and Gram-positive bacteria. Gram-negative bacteria were resistant to tolaasin, but became susceptible when treated concomitantly with sub-minimal inhibitory concentrations of polymyxin B. P. tolaasii was resistant to tolaasin even in the presence of polymyxin B.

The biological activities of the lipodepsipeptides (LDP) white line–inducing principle (WLIP), produced by Pseudomonas reactans NCPPB1311, and tolaasin I, produced by P. tolaasii NCPPB2192, were compared. Antimicrobial assays showed that both LDP inhibited the growth of fungi—including the cultivated mushrooms Agaricus bisporus, Lentinus edodes, and Pleurotus spp.—chromista, and gram-positive bacteria. Assays of the two LDP on blocks of Agaricus bisporus showed their capacity to alter the mushrooms’ pseudo-tissues though WLIP was less active than that of tolaasin I. Contrary to previous studies, tolaasin I was found to inhibit the growth of gram-negative bacteria belonging to the genera Escherichia, Erwinia, Agrobacterium, Pseudomonas, and Xanthomonas. The only gram-negative bacterium affected by WLIP was Erwinia carotovora subsp. carotovora. Both WLIP and tolaasin I caused red blood cell lysis through a colloid-osmotic shock mediated by transmembrane pores; however, the haemolytic activity of WLIP was greater than that of tolaasin I. Transmembrane pores, at a concentration corresponding to 1.5 × C50, showed a radius between 1.5 and 1.7 ± 0.1 nm for WLIP and 2.1 ± 0.1 nm for tolaasin I. The antifungal activity of WLIP together with the finding that avirulent morphological variants of P. reactans lack WLIP production suggests that WLIP may play an important role in the interaction of the producing bacterium P. reactans and cultivated mushrooms. Additional keywords: Pseudomonas tolaasii.

Tolaasin is a peptide toxin produced by Pseudomonas tolaasii and causes brown blotch disease of the cultivated mushrooms. Two types of ion channels were identified by the incorporation of tolaasin into lipid bilayer. The slope conductance of type 1 channel measured in the buffer containing 100 mM KCl was 150 pS with a linear current vs. voltage relationship. The type 2 tolaasin channel had two subconductance states of 300 and 500 pS. Both channels were inhibited by Zn(2+). Ion channel formations of tolaasin were concentration-dependent and single channel currents were successfully obtained at 0.6 unit tolaasin, 15.9 nM. The type 1 channel was obtained more frequently than the type 2 channel and the ratio of their appearance was approximately 4:1, respectively.

Shaken culture in KB of strain NCPPB2192 of Pseudomonas tolaasii showed an activity of 3,200 and 800 units ml−1 toward Bacillus megaterium and Rhodotorula pilimanae, respectively. On the contrary, an activity of 400 units ml−1 toward B. megaterium was observed with the cultures of strain NCPPB1311 of P. “reactans”. The purification of lipodepsipeptides from the cultures of P. tolaasii and P. “reactans” yielded about 13 mg l−1 of tolaasin and 169 mg l−1 of White Line Inducing Principle (WLIP), respectively. Antimicrobial assays showed that HPLC grade tolaasin I inhibited the growth of filamentous fungi and yeasts and Gram positive bacteria. Only a limited activity was observed toward some Gram negative bacteria. Of particular interest is the activity of tolaasin I toward phytopathogenic fungi, Pleurotus and Agaricus spp., as well as yeasts and filamentous fungi responsible for mycoses of mammals. WLIP inhibited the growth of fungi and Gram positive bacteria, though at higher concentration when compared to tolaasin I, but not that of Gram negative bacteria. Assays of the two toxins on blocks of A. bisporus confirmed the antifungal activity of the two lipodepsipeptides and suggested their possible role in the symptoms caused by the mushroom bacterial pathogens.

Infection of Agaricus bisporus with Pseudomonas tolaasii or treatment with a tolaasin containing preparation resulted in the activation of tyrosinase and the development of a brown discolouration of the fruitbody. In order to investigate whether tolaasin is responsible for these reactions, mushrooms were treated with different concentrations of HPLC-purified tolaasin. Tolaasin was found to induce both browning and the activation of tyrosinase, with the level of activation being related to the concentration of toxin applied. In water-treated controls, active tyrosinase isoforms with pIs of 4.5 and 4.4 were the main isoforms found but tolaasin treatment resulted in the activation of a tyrosinase isoform with a pI of 5.7. The different active isoforms could be distinguished as the tolaasin activated isoforms had a higher affinity for catechol as a substrate (Km0.82 m m ) than the active isoforms in the control (Km2.86 m m ). In mushrooms inoculated with P. tolaasii or treated with tolaasin containing extracts no significant differences were found in the mechanism of oxidation of tyrosine, γ-glutaminyl-4-hydroxybenzene or γ-glutaminyl-3,4-dihydroxybenzene to form melanin. In tolaasin treated samples, a constant total amount of melanin and sum of the three phenols was found.

Pseudomonas tolaasii is a bacterium endemic to the compost beds where common mushroom (Agaricus bisporus) is cultivated. Under some environmental conditions still not well-determined, but influenced by temperature and relative humidity, the bacterium can become pathogenic and provoke the brown blotch disease. This review describes the interaction between P. tolaasii and A. bisporus that results in the appearance of brown spots on the mushroom caps, typical symptoms of the disease. Firstly, P. tolaasii is studied, the changes in pathogenicity are explained, the compounds that provoke the damage are enumerated as well as various experimental methods to identify the pathogenic form of the bacteria. Secondly, mechanisms involved in the formation of the brown colour on the A. bisporus caps upon infection are briefly mentioned, taking into account the enzymes that catalyse the reaction, their mechanism, substrates and reaction products. Afterwards, a detailed description of the infection process is presented step by step, starting by the chemotactical attraction, fixation, secretion of the toxins, membrane breakdown, effect of the toxin on mushroom polyphenol oxidases and on the discolouration reaction. A possible mechanism of infection is hypothesised at the molecular level. Finally, the strategies tested until now to control the disease are discussed.

Pseudomonas tolaasii, the pathogen of brown blotch disease in a cultivated mushroom, Pleurotus ostreatus, produces extracellular toxins (tolaasins) that are the major cause of symptoms. Components of the fruiting bodies activate tolaasin production and suppress the occurrence of avirulent variants of the bacterium. Single mini-Tn5km1 insertions in P. tolaasii strain PT814 yielded mutants that are avirulent to P. ostreatus but weakly virulent to Agaricus bisporus, causing yellow blotch rather than typical brown blotch. Mutants are pleiotropically deficient in tolaasin as well as protease production, but sustain a normal level of volatile toxin (tovsin) in vitro. These characteristics resemble variants from a genetically unstable P. tolaasii strain, S8501. The rtpA gene required for tolaasin production in the pleiotropic mutants was cloned. Nucleotide sequence predicted that RtpA has a striking homology to the BvgS family of bacterial two-component regulators. Observations suggest that signals from hosts stimulate the production of factors important to virulence of P. tolaasii.

Three A. bisporus strains (U1, C45 and B62) with different resistance towards bacterial blotch were infected with Pseudomonas tolaasii or with a partly purified toxin extract. In the two susceptible strains (U1 and B62), the total tyrosinase level decreased upon treatment with bacteria or the toxin-containing extract. The amount of active tyrosinase increased, suggesting an activation of tyrosinase upon infection. In the resistant strain (C45), the tyrosinase activity remained unchanged. Protease levels were found to be increased in regions of the sporocorp that were infected with the pathogenic bacterium, but not when treated with the toxin preparation, suggesting an activation of tyrosinase by a non-proteolytic mechanism. This observation was confirmed with in vitro experiments in which tyrosinase was found to be activated by a protease-free tolaasin-containing preparation.