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

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

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.     

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.     

Pseudomonas tolaasii on mushroom (Agaricus bisporus) crops: bactericidal effects of six disinfectants and their toxicity to mushrooms

N -Cetylpyridinium chloride, benzalkonium chloride, Cetrimide, bronopol (2-bromo-2-nitropropane-1,3-diol), Panacide and Chloramine T were tested as possible disinfectants for use in growing mushrooms (Agaricus bisporus) where Pseudomonas tolaasii blotch is prevalent. The most effective materials in vitro against Ps. tolaasii where the quaternary ammonium compounds and bronopol in terms of the MIC and MCC tests. In 8 min 'clean' and 'dirty' tests incorporating yeast cells bronopol did not kill the pathogen, whereas the other five disinfectants did so. If mushroom casing (peat plus limestone) was added to these short duration tests the pathogen survived all six disinfectants. When tests with added casing were extended to 20 h, bronopol was very effective (cidal value 100 µg/ml) and the pathogen was not killed by the other five disinfectants. In experiments on agar plates, bronopol and chloramine T were stimulating to the growth of A. bisporus. Growing mushroom caps treated with bronopol remained white, whereas caps treated with the other five disinfectants turned brown within 30 min. It is thus likely that bronopol could be used to control the source of bacterial blotch epidemics in mushroom growing, which previous work has shown to be in the casing.     

Pseudomonas tolaasii in cultivated mushroom (Agaricus bisporus) crops: numbers of the bacterium and symptom development on mushrooms grown in various environments after artificial inoculation

The recovery of Pseudomonas tolaasii applied to peat, limestone and mushroom caps, is very difficult, recovery rates being 0.2–16.0%. Without Agaricus bisporus mycelium, inoculated Ps.tolaasii disappears in the casing layer. As mushroom primordia grew in size on inoculated mushroom beds, the number of detectable cells of the pathogen increased. Symptoms of blotch disease became visible when 5.4 times 10⁶ cfu were detectable, when the mushroom primordia were 6 mm in diameter; 60% of mushrooms showed symptoms before they were 15 mm in diameter. Application of Ps.tolaasii cells as low as 20 cfu/cm² of bed gave epidemics of this severity. Neither size nor age of mushrooms affects their susceptibility. When Ps.tolaasii was placed directly onto caps, 6 times 10⁷ cfu were necessary to produce a blotch lesion (though only 3.5 times 10⁶ cfu could be recovered). Changes in r.h. and temperature did not affect the numbers of cells of Ps.tolaasii on inoculated caps; very frequent watering did so. Increased severity of the disease was seen only on over-watered mushrooms; this occurred by increase in the size of lesions seen at the primordium stage. The number of cells of Ps.tolaasii present on the early primordial stages of mushroom growth controls the extent of blotch disease seen at harvesting, whereas variations in r.h. or temperature during growing do not do so. An illustrated disease symptom measurement key (of general application for assessing severity of blotch disease) is included in the text.     

Pseudomonas tolaasii control by kasugamycin in cultivated mushrooms (Agaricus bisporus)

Pseudomonas tolaasii , causing brown blotch disease on the edible mushroom Agaricus bisporus , was effectively controlled by kasugamycin. An artificial infection was first established in the first flush, by inoculating the button-sized mushrooms of the first flush with a suspension of Ps. tolaasii. A 1% aqueous solution of kasugamycin supplied on the button-sized mushrooms of the second flush drastically reduced bacterial blotch symptoms on these mushrooms at picking stage. Disease incidence in the second flush in the control treatment (inoculated with Ps. tolaasii ) was composed of 18% lightly, 29% moderately and 10% heavily affected mushrooms, which totalled up to 57% affected. The 1% kasugamycin treatment significantly reduced total disease incidence to only 9% (lightly) affected. Single sodium hypochlorite treatments showed no result.     

Left handed alpha-helix formation by a bacterial peptide

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

Phenotypic variation of Pseudomonas putida and P. tolaasii affects the chemotactic response to Agaricus bisporus mycelial exudate.

The chemotactic response of wild-type Pseudomonas putida and P. tolaasii, and a phenotypic variant of each species, to Agaricus bisporus mycelial exudate was examined. Both P. putida, the bacterium responsible for initiating basidiome development of A. bisporus, and P. tolaasii, the causal organism of bacterial blotch disease of the mushroom, displayed a positive chemotactic response to Casamino acids and to A. bisporus mycelial exudate. The response was both dose- and time-dependent and marked differences were observed between the response time of the wild-type strains and their phenotypic variants. Phenotypic variants responded rapidly to both attractants and reached a maximum response after 10-20 min, whereas the wild-types took 45-60 min. The differences are partly explained by the more rapid swimming speed of the phenotypic variants. Both variants responded maximally to similar concentrations of Casamino acids and mycelial exudates. Investigations into the nature of the attractants contained in the mycelial exudate indicated that they are predominantly small (Mr less than 2000) thermostable compounds. Sugars present in the exudate did not elicit a chemotactic response in any isolate, but a mixture of 14 amino acids detected in the exudate accounted for between 50 and 75% of the chemotactic response of the fungal exudate.     

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.     

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.     

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.     

Identification of exopolysaccharides produced by fluorescent pseudomonads associated with commercial mushroom (Agaricus bisporus) production.

The acidic exopolysaccharides (EPSs) from 63 strains of mushroom production-associated fluorescent pseudomonads which were mucoid on Pseudomonas agar F medium (PAF) were isolated, partially purified, and characterized. The strains were originally isolated from discolored lesion which developed postharvest on mushroom (Agaricus bisporus) caps or from commercial lots of mushroom casing medium. An acidic galactoglucan, previously named marginalan, was produced by mucoid strains of the saprophyte Pseudomonas putida and the majority of mucoid strains of saprophytic P. fluorescens (biovars III and V) isolated from casing medium. One biovar II strain (J1) of P. fluorescens produced alginate, a copolymer of mannuronic and guluronic acids, and one strain (H13) produced an apparently unique EPS containing neutral and amino sugars. Of 10 strains of the pathogen "P. gingeri," the causal agent of mushroom ginger blotch, 8 gave mucoid growth on PAF. The "P. gingeri" EPS also was unique in containing both neutral sugar and glucuronic acid. Mucoid, weakly virulent strains of "P. reactans" produced either alginate or marginalan. All 10 strains of the pathogen P. tolaasii, the causal agent of brown blotch of mushrooms were nonnmucoid on PAF. Production of EPS by these 10 strains plus the 2 nonmucoid strains of "P. gingeri" also was negative on several additional solid media as well as in two broth media tested. The results support our previous studies indicating that fluorescent pseudomonads are a rich source of novel EPSs.     

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.     

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

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

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

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

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.     

Brown Discolouration of Mushrooms Caused by Pseudomonas agarici

Incidence of brown-discoloured mushrooms, differing from brown blotch disease caused by Pseudomonas tolaasii , has increased in the Netherlands and been responsible for considerable economic losses. A comparative SEM-study of diseased tissue revealed that hyphae were collapsed and covered with a plaque in which numerous bacteria were embedded. The predominant bacterium identified was Pseudomonas agarici. Both our isolated P. agarici strain and the type strain P. agarici LMG 2112 caused brown discolouration of mushrooms after spraying with bacterial suspensions. Drippy-gill symptoms were not observed.     

Interaction between gfp-tagged Pseudomonas tolaasii P12 and Pleurotus eryngii

Pseudomonas sp., (formerly reported as strain P12) which produces brown blotch disease symptoms on Pleurotus eryngii, has been identified as P. tolaasii based on its biochemical, physiological properties and 16S rDNA sequence analysis. This pathogen is able to infect basidiocarps when surface-inoculated on mushroom casing soil. However, infected basidiocarps develop the brown blotch disease symptoms when the pathogen concentration in the fruiting body tissues is higher than 10(4) cfu/g d.w. Using gfp-tagged cells and confocal laser scanning microscopy, it was possible to show that the pathogen has the ability to tightly attach to the hyphae of Pleurotus eryngii.     

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

Tolaasin, a pore-forming peptide toxin, is produced by Pseudomonas tolaasii and causes brown blotch disease of the cultivated mushrooms. P. tolaasii 6264 was isolated from the oyster mushroom damaged by the disease in Korean. In order to isolate tolaasin molecules, the supernatant of bacterial culture was harvested at the stationary phase of growth. Tolaasin was prepared by ammonium sulfate precipitation and three steps of chromatograpies, including a gel permeation and two ion exchange chromatographies. Specific hemolytic activity of tolaasin was increased from 1.7 to 162.0 HU mg(-1) protein, a 98-fold increase, and the purification yield was 16.3%. Tolaasin preparation obtained at each purification step was analyzed by HPLC and SDS-PAGE. Two major peptides were detected from all chromatographic preparations. Their molecular masses were analyzed by MALDI-TOF mass spectrometry and they were identified as tolaasin I and tolaasin II. These results demonstrate that the method used in this study is simple, time-saving, and successful for the preparation of tolaasin.     

The enigma of double-stranded RNA (dsRNA) associated with mushroom virus X (MVX)

New variants of pathogenic fungal viruses are emerging and they are enigmatic in revealing their molecular identity and of their origin. Double-stranded RNAs, some in non-encapsidated forms are increasingly becoming causal agents for sporadic diseases and are consistently associated with a complex profile of dsRNAs, presumably of (multiple) viral origin present in the host while the same are conspicuously absent in healthy (looking) counterparts. The emergence of an unusual Agaricus bisporus mushroom 'patch disease' first reported in 1996, later termed as 'mushroom virus X' (MVX), exhibited a wide range of symptoms (e.g. barren patches beside healthy looking mushrooms, arrested pins, premature opening, brown, off-colour and distortions in shape). A variable compendium of novel 26 (dsRNA) elements, ranging in sizes between 20.2kb to 0.64kb, several of them (-17/26) in non-encapsidated form have been shown to occur in the diseased mushroom fruiting bodies and are thought to comprise multiple viruses. Ten years on, this devastating disease is now more widespread and prevalent in a number of European countries (e.g. The Netherlands, Ireland) ranging from occasional to severe outbreaks leading to crop losses. Impressive data has been accumulated on the symptoms, but the potential aetiological sources, biochemical and molecular characterizations corresponding to the symptoms vis-a-vis MVX linked dsRNAs still remain either elusive or unclear. We have overviewed mainly the molecular findings of research groups working on MVX in these countries together with our own work on MVX in Northern Ireland. To date, the results reviewed suggest that with the exception of 4 low molecular weight dsRNA bands (sizes 2.0. 1.8, 0.8 and 0.6 kb) which consistently were found synchronous to mushroom off-colour/browning symptoms in the UK and The Netherlands, other individual MVX dsRNAs or their banding patterns clearly lack credible relationship with other symptoms of the MVX disease complex. The issues in the molecular characterisation of the MVX dsRNAs include the disparate results on the molecular sequences obtained for some of these by the different research groups, the varying molecular methods or approaches adopted by them for deciphering the nucleotide sequences of the novel dsRNAs that are different from previously encountered mushroom viruses. The future outlook and general consensus among mushroom researchers worldwide is for an urgent need to recruit international taskforce and re-focus on clarifying the symptom vis-a-vis dsRNAs in the enigmatic MVX disease complex. As crossing the cellular membrane is a key step to infection process, we have also attempted to draw parallels with other viruses in terms of the potential cell entry mechanisms for MVX dsRNAs. In the light of MVX disease and A. bisporus being a commercial crop worldwide in agri-food markets, and taking cue from its nearest Basidiomycete model mushroom, Coprinus cinereus whose genome mapping is completed, we also propose that it may be timely for the international research groups to renew efforts to prop up a network for sequencing the host A. bisporus mushroom genome (-38 MB) for a better understanding of host-pathogen relationships.