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

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

Identification and characterization of a locus which regulates multiple functions in Pseudomonas tolaasii, the cause of brown blotch disease of Agaricus bisporus.

Pseudomonas tolaasii, the causal agent of brown blotch disease of Agaricus bisporus, spontaneously gives rise to morphologically distinct stable sectors, referred to as the phenotypic variant form, at the margins of the wild-type colonies. The phenotypic variant form is nonpathogenic and differs from the wild type in a range of biochemical and physiological characteristics. A genomic cosmid clone (pSISG29) from a wild-type P. tolaasii library was shown to be capable of restoring a range of characteristics of the phenotypic variant to those of the wild-type form, when present in trans. Subcloning and saturation mutagenesis analysis with Tn5lacZ localized a 3.0-kb region from pSISG29, designated the pheN locus, required for complementation of the phenotypic variant to the wild-type form. Marker exchange of the Tn5lacZ-mutagenized copy of the pheN locus into the wild-type strain demonstrated that a functional copy of the pheN gene is required to maintain the wild-type pathogenic phenotype and that loss of the pheN gene or its function results in conversion of the wild-type form to the phenotypic variant form. The pheN locus contained a 2,727-bp open reading frame encoding an 83-kDa protein. The predicted amino acid sequence of the PheN protein showed homology to the sensor and regulator domains of the conserved family of two component bacterial sensor regulator proteins. Southern hybridization analysis of pheN genes from the wild type and the phenotypic variant form revealed that DNA rearrangement occurs within the pheN locus during phenotypic variation. Analysis of pheN expression with a pheN::lacZ fusion demonstrated that expression is regulated by environmental factors. These results are related to a model for control for phenotypic variation in P. tolaasii.     

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

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

A note on ginger blotch, a new bacterial disease of the cultivated mushroom, Agaricus bisporus

Ginger blotch, a new bacterial disease of the cultivated mushroom, Agaricus bisporus , is described from farms in the UK. The symptoms are distinct from the classical blotch disease caused by Pseudomonas tolaasii. The causative organism has been isolated and identified as a new member of the Pseudomonas fluorescens complex which can be distinguished from Pseudomonas tolaasii by several simple tests.     

Bacterial blotch disease of the cultivated mushroom Agaricus bisporus: screening, isolation and characterization of bacteria antagonistic to the pathogen (Pseudomonas tolaasii)

Bacteria, mainly pseudomonads, were isolated from mushroom farms and from soil and plant materials. They were screened for antagonism to Pseudomonas tolaasii , the cause of bacterial blotch of mushroom, using an exclusion zone assay against a bacterial lawn of the pathogen. Selected potential antagonists were identified by the API system and whole cell fatty acid profiles. These strains were tested further in the white line test and host pathogenicity test with mushroom caps. Some of the antagonists have been stable in their aggressiveness over 1 year and several transfers during storage on nutrient agar.     

Pseudomonas tolaasii in cultivated mushroom (Agaricus bisporus) crops: effects of sodium hypochlorite on the bacterium and on blotch disease severity

Sodium hypochlorite killed Pseudomonas tolaasii in water in 30 s at pH 6.0 when 5 mg/1 free available chlorine (FAC) was used. On glass beads 62.5 mg/1 FAC was necessary to kill the pathogen in 30 s. Peat and limestone mixture ('casing') prevented some cells of the pathogen being killed by chlorine. Casing treated with 50 and 100 mg/1 FAC still contained some Ps. tolaasii cells which were later able to multiply. Although some viable cells of the pathogen survived the use of 150 mg/1 FAC these were apparently unable to multiply. Mushroom tissue is more 'disinfectant-wasting' than casing, the pathogen on it surviving 250 mg/1 FAC for 10 min. In controlled environmental experiments, use of 150 mg/1 FAC at mushroom 'pinning' (2.5 mm diameter primordia) gave as much control of blotch disease as was obtainable if chlorination began after casing. Delay in starting chlorination until the mushrooms were 10 to 15 mm in diameter resulted in blotch disease incidence and severity as severe as in unchlorinated controls. Disease incidence was not reduced when 50, 100 and 150 mg/1 FAC was used, but disease severity was significantly reduced when 150 mg/1 was used. Adjusting the pH of the water did not affect these results. On commercial farms, routine watering with 150 mg/1 FAC starting at pinning, checked frequently by the sodium arsenite titrimetric method, for 3 years, reduced the percentage of mushrooms discarded because of very severe Ps. tolaasii blotch from 5.2% to 0.6% on one farm and from 7.4% to 0.5% on another, but did not eliminate the disease completely.     

Characterization by 16S rRNA sequence analysis of pseudomonads causing blotch disease of cultivated Agaricus bisporus

Bacterial blotch of Agaricus bisporus has typically been identified as being caused by either Pseudomonas tolaasii (brown blotch) or Pseudomonas gingeri (ginger blotch). To address the relatedness of pseudomonads able to induce blotch, a pilot study was initiated in which pseudomonads were selectively isolated from mushroom farms throughout New Zealand. Thirty-three pseudomonad isolates were identified as being capable of causing different degrees of discoloration (separable into nine categories) of A. bisporus tissue in a bioassay. These isolates were also identified as unique using repetitive extragenic palindromic PCR and biochemical analysis. Relationships between these 33 blotch-causing organisms (BCO) and a further 22 selected pseudomonad species were inferred by phylogenetic analyses of near-full-length 16S rRNA gene nucleotide sequences. The 33 BCO isolates were observed to be distributed throughout the Pseudomonas fluorescens intrageneric cluster. These results show that in addition to known BCO (P. tolaasii, P. gingeri, and Pseudomonas reactans), a number of diverse pseudomonad species also have the ability to cause blotch diseases with various discolorations. Furthermore, observation of ginger blotch discoloration of A. bisporus being independently caused by many different pseudomonad species impacts on the homogeneity and classification of the previously described P. gingeri.     

Isolation of a gram-positive bacterium effective in suppression of brown blotch disease of cultivated mushrooms,Pleurotus ostreatus andAgaricus bisporus, caused byPseudomonas tolaasii

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., MicroStation^TMSystem 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.     

PCR assays for specific and sensitive detection of Pseudomonas tolaasii,the cause of brown blotch disease of mushrooms

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.     

Adapting substrate formulas used for shiitake for production of brown Agaricus bisporus

A pasteurized, non-composted substrate (basal mixture) consisting of oak sawdust (28%), millet (29%), rye (8%), peat (8%), alfalfa meal (4%), soybean flour (4%), wheat bran (9%), and CaCO3 (10%) was adapted from shiitake culture to produce the common cultivated mushroom (brown; portabello), Agaricus bisporus. Percentage biological efficiency (ratio of fresh mushroom harvested/oven-dry substrate weight, %BE) ranged from a low of 30.1% (when wheat straw was substituted for sawdust) to 77.1% for the basal mixture. Special, high gas-exchange bags were required to optimize mycelial growth during spawn run. Our formula may allow specialty mushroom growers to produce portabello mushrooms on a modified, pasteurized (110 degrees C for 20 min) substrate commonly used for shiitake production without the added expense of compost preparation.     

Response of Pseudomonas tolaasii,the causal agent of mushroom brown blotch disease to the volatile compounds produced by endofungal bacteria

Volatile organic compounds (VOCs) produced by bacteria have significant potential to control phytopathogens. In this study, the VOCs produced by endofungal bacteria Pseudomonas sp. Bi1, Bacillus sp. De3, Pantoea sp. Ma3 and Pseudomonas sp. De1 isolated from wild growing mushrooms were evaluated in vitro for their antagonistic activity against Pseudomonas tolaasii Pt18, the causal agent of mushroom brown blotch disease. The gas chromatography–mass spectrometry (GC–MS) analysis revealed that strains Pseudomonas sp. Bi1, Pseudomonas sp. De1, Bacillus sp. De3 and Pantoea sp. Ma3 produced eight, sixteen, nine, and twelve VOCs, respectively. All antagonistic endofungal bacteria produced VOCs which significantly reduced brown blotch symptoms on mushroom caps and inhibited the growth of P. tolaasii Pt18 at the varying levels. Scanning electron microscopy revealed severe morphological changes in cells of P. tolaasii Pt18 following exposure to the VOCs of Pseudomonas sp. Bi1 and De1. Furthermore, The VOCs produced by endofungal bacteria significantly reduced swarming, swimming, twitching, chemotaxis motility and biofilm formation by P. tolaasii Pt18 cells, which are essential contributors to pathogenicity. This is to first report about the inhibition effects of VOCs produced by antagonistic bacteria on virulence traits of P. tolaasii. Our findings provide new insights regarding the potential of antibacterial VOCs as a safe fumigant to control mushroom brown blotch disease.

     

Submerged growth of the cultivated mushroom,Agaricus bisporus

Agaricus bisporus grew well in submerged culture in a medium containing malt extract, phosphate, and casein. Moderate growth occurred in defined media containing glucose, asparagine, phenylalanine, vitamins and minerals. Other amino acids did not stimulate growth. Growth was stimulated by vegetable oils, partly due to utilization of the oils, and partly to a more complex mechanism. Oleates had the same effect as vegetable oils; palmitates a lesser one. In shaken flasks maximum yield was reached after 22–24 days and in stirred and aerated fermentors after 8–10 days. Besides dry weight of mycelium, laccase activity was determined. The latter determination is suitable for a rapid estimation of the growth in routine experiments. The flavour of the mycelium was like that of mushrooms but weaker. It was strongest in standing liquid cultures and on solid media. The mycelium grown in submerged culture was suitable as spawn for mushroom culture. Presented at the First International Mycological Congress, Exeter, 7–16 September 1971, and at the Meeting of the Netherlands Society for Microbiology, Rotterdam, 8 December 1971. We thank the Mushroom Experiment Station, Horst, the Netherlands, for kindly supplying the compost for fructification experiments and Mr. P. Arntz, M.Sc., for advice in the fermentor work. F. IJ. Dijkstra is indebted to the Royal Netherlands Fermentation Industries (Gist-Brocades), Delft, for a research grant.     

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

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

Biological control of mushroom brown blotch disease using antagonistic bacteria

Button mushroom brown blotch disease is one of the most important and devastating diseases in Iran which is caused by Pseudomonas tolaasii. To screen antagonistic bacteria against this pathogen, major mushroom cultivation centres in Iran were surveyed and samples were taken from compost, soil cover and button caps with or without visible symptoms. In total, 120 bacteria were isolated on the basis of their morphology and pathology on excised tissue blocks of the fresh Agaricus bisporus. Among all tested bacteria, thirty-six isolates produced variable inhibition zones and reduced the symptom incidence on tissue blocks of A. bisporus. The most effective antagonistic bacteria caused almost 100% inhibition of pathogenic bacterium. To confirm the identification of antagonistic bacteria, RNA polymerase beta-subunit gene (rpoB) of five antagonistic strains (A1, A2, A3, A4 and A6) were amplified using primer sets of long amplicon primers (LAPS) and LAP27. The polymerase chain reaction products of the strains A1, A3 and A6 were sequenced. Based on phenotypic, biochemical and molecular characteristics, the bacterial antagonists were identified as P. putida (A1), P. reactants (A2 and A6), P. fluorescens (A3 and A4) and Bacillus subtilis (A5), respectively. In all four criteria including weight of the diseased and healthy caps, per cent disease severity and per cent disease incidence, the treatment 2 (P. fluorescens A4) was the most effective from among T1, T3, T4 and T5 treatments. Overall results of this study suggest that bacterial antagonists may be potential biocontrol agents for biological promotion of the health and growth of button mushroom.     

Protective effect of a protease inhibitor from Agaricus bisporus on Saccharomyces cerevisiae cells against oxidative stress

Abstract

Protease inhibitors are known to resist damage to host organisms against external threats, hence form a part of their defense system. This property of protease inhibitors was studied on protecting oxidatively stressed Saccharomyces cerevisiae yeast cells. The protease inhibitor was extracted from Agaricus bisporus, an edible mushroom. The inhibitor showed the presence of antioxidant activity as the purified inhibitor fraction gave an IC₅₀ value of 45.13?±?0.88?µg/mL and 33.30?±?1.5?µg/mL when checked, respectively, by 2, 2-diphenyl-1-picrylhydrazyl, DPPH and 2, 2′-azo-bis(3-ethylbenzthiazoline-6- sulfonic acid), ABTS?+ scavenging activity. The yeast cells’ survival rate (%), was determined through 3-(4, 5-dimethylthiazol-2-yl) - 2, 5-diphenyltetrazolium bromide, MTT assay, and it was found that in the presence of 2?mM H₂O₂ cell survival decreased to 26.33%, whereas when the experiment was conducted in the presence of protease inhibitor and 2?mM H₂O₂ cell survival percentage rose to 74%. The protease inhibitor’s effect on the oxidatively stressed yeast cells was further studied by using Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM) and Confocal Microscopy to understand the morphological changes. The viable and non-viable cell populations were quantified using Fluorescence Assorted Cell Sorting (FACS) using propidium iodide, PI, 4′, 6-diamidino-2-phenylindole, DAPI and 2′, 7′-dichlorofluorescein, DCF dyes.