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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.

Among the biotic constraints of common mushroom (Agaricus bisporus) production, bacterial blotch is considered the most important mushroom disease in terms of global prevalence and economic impact. Etiology and management of bacterial blotch has been a major concern since its original description in 1915. Although Pseudomonas tolaasii is thought to be the main causal agent, various Pseudomonas species, as well as organisms from other genera have been reported to cause blotch symptoms on mushroom caps. In this review, we provide an updated overview on the etiology, epidemiology, and management strategies of bacterial blotch disease. First, diversity of the causal agent(s) and utility of high throughput sequencing-based approaches in the precise characterization and identification of blotch pathogen(s) is explained. Further, due to the limited options for use of conventional pesticides in mushroom farms against blotch pathogen(s), we highlight the role of balanced threshold of relative humidity and temperature in mushroom farms to combat the disease in organic and conventional production. Additionally, we discuss the possibility of the use of biological control agents (either antagonistic mushroom-associated bacterial strains or bacteriophages) for blotch management as one of the sustainable approaches for 21stcentury agriculture. Finally, we aim to elucidate the association of mushroom microbiome in cap development and productivity on one hand, and blotch incidence/outbreaks on the other hand.

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.

톨라신은 Pseudomonas tolaasii에 의해 생성되는 펩티드 독소이며, 인공재배 버섯에 갈반병을 일으킨다. 톨라신 펩티드는 막에 pore를 형성하고, 세포막 구조를 파괴한다. 톨라신의 분자 작용은 톨라신 분자들의 응집, 세포막 결합, 세포막에서의 pore 형성으로 이루어져 있다. 따라서, 이 작용의 억제는 갈반병을 억제할 수 있다. 식품첨가물로부터 몇몇의 톨라신 저해제(tolaasin inhibitory factors, TIF)를 얻었다. TIF는 버섯에 감염된 병원균에 의한 갈반 형성을 억제할 수 있었다. 본 연구에서는 TIF를 다양한 조건에서 보관하였으며, 톨라신에 의한 세포 독성의 저해에 대해 이들의 활성을 조사하였다. TIF는 불포화탄소 화합물이기 때문에 대기 노출과 빛 조사에 민감하다. 혐기적 조건에서 TIF는 3개월동안 안정하였고, 효과는 10% 정도 감소하였다. 그러나 빛과 공기와 접촉한 조건에서는 2주동안 90% 가까이 TIF 활성이 억제되었다. 5, 25, 45 ℃의 저장온도에서는 어떠한 차이도 보이지 않았기 때문에 온도는 TIF의 안정성에 유의적인 영향을 끼치지 않았다. 따라서, TIF 화합물의 안정적인 저장을 위해서는 용기는 밀폐되고, 빛은 차단되어야 한다.

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.

Tolaasin은 Pseudomonas tolaasii에 의해 생성되어 pore를 형성 하는 펩티드 독소이며, 인공재배 버섯의 막 구조를 파괴하여 갈 반병을 일으킨다. Tolaasin이 막에서 pore를 형성하는 기작이나 특성은 자세히 알려지지 않았으나, 인공 지질이중막에서 tolaasin 에 의한 pore 형성은 제시되었다. Tolaasin에 의한 지질막에서의 pore 형성은 드물게 나타났고, 형성된 pore는 불안정하기에 tolaasin pore의 길이와 지질막의 두께가 서로 일치하지 않을 수 있음이 제안되었다. 그러므로, 탄소수가 다른 지방산으로 이루어 진 인지질들을 첨가하여 tolaasin에 의한 용혈활성 변화를 측정하였다. 두 개의 decanoic acids (C10:0, 1,2-didecanoyl-sn-glycero- 3-phosphoethanolamine; DDPE)와 myristic acids (C14:0, 1,2- dimyristoyl-sn-glycero-3-phosphoethanolamine), stearic acids (C18:0, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine)로 이루 어진 phosphatidylethanolamine들을 적혈구와 tolaasin 펩티드가 포함된 반응용액에 가했을 때, DDPE만이 tolaasin에 의한 용혈 활성을 촉진하였으며, 나머지 두 인지질은 효과를 보이지 않았다. DDPE를 다양한 농도로 처리하였을 때, tolaasin에 의한 용혈활성은 농도의존적으로 증가하였다. 중간길이의 지방산으로 구성된 인지질은 tolaasin pore 구조와 막지질 사이에 결합하여 pore 주변의 막을 얇게 함으로써 tolaasin pore를 안정화시킬 것으로 여겨진다. 본 연구의 결과는 중간 크기의 지방산으로 구 성된 인지질이 tolaasin pore를 막 구조에서 안정화시킴으로써 활성을 증가시키며, 이것은 적혈구 막에서 tolaasin pore의 길이 가 막의 두께보다 조금 짧을 것이라는 사실을 제안한다.

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.

Tolaasin, a pore-forming peptide toxin produced by Pseudomonas tolaasii, causes brown blotch disease on cultivated mushrooms. Hemolysis using red blood cells was measured to evaluate the cytotoxicity of tolaasin. To investigate the mechanism of tolaasin-induced cell disruption, we studied the effect of temperature on the hemolytic process. At 4 degrees Celsius, poor binding of the tolaasin molecules to the erythrocyte membrane was observed and most of the tolaasin molecules stayed in the solution. However, once tolaasin bound to erythrocytes at 37 degrees Celsius and the temperature was decreased, complete hemolysis was observed even at 4 degrees Celsius. These results indicate that tolaasin binding to cell membrane is temperature-sensitive while tolaasin-induced membrane disruption is less sensitive to temperature change. The effect of erythrocyte concentration was measured to understand the membrane binding and pore-forming properties of tolaasin. The percentage of hemolysis measured by both hemoglobin release and cell lysis decreased as erythrocyte concentration increased in the presence of a fixed amount of tolaasin. The result shows that hemolysis is dependent on the amount of tolaasin and multiple binding of tolaasin is required for the hemolysis of a single cell. In analysis of dose-dependence, the hemolysis was proportional to the tenth power of the amount of tolaasin, implying that tolaasin-induced hemolysis can be explained by a multi-hit model.

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 and , we found that is another antagonist to block the toxicity of tolaasin. inhibited the tolaasin-induced hemolysis in a dose-dependent manner and its Ki value was mM, implying that the inhibitory effect of is stronger than that of . The hemolytic activity was completely inhibited by at the concentration higher than 50 mM. The effect of was reversible since it was removed by the addition of EDTA. When the tolaasin-induced hemolysis was suppressed by the addition of 20 mM , the subsequent addition of EDIA immediately initiated the hemolysis. Although the mechanism of -induced inhibition on tolaasin toxicity is not known, 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 inhibits the pore activity of tolaasin, the last step of the toxic process.

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.

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.

Discolouration due to senescence differs from blotch-related discolouration, which might indicate differential mechanisms being operational. In samples infected with bacteria or with a partially purified toxin extract, a higher degradation of total tyrosinase than in senescening mushrooms was found. Simultaneously, the active tyrosinase was increasing resulting in an increase in percentage of active tyrosinase. Phenolic substrates of the active tyrosinase were being oxidized, proportionally to the damage detectable on the mushroom cap. γ-Glutaminyl-3,4-dihydroxybenzene was degraded first, followed by γ-glutaminyl-4-hydroxybenzene and later tyrosine. The amount of melanin that was synthesized was larger than the sum of oxidation of the phenols measured. Principal Component Analysis explained 84% of the variance in symptoms, and it demonstrated the phenol oxidation and active tyrosinase level as the most important parameters for the browning induced by bacteria or a tolaasin preparation treatment.