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 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.     

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.     

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.     

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.     

Genetic diversity in pseudomonads associated with cereal cultures infected with basal bacteriosis

The genetic properties of 45 pseudomonad strains isolated from cereal cultures exhibiting symptoms of basal bacteriosis have been investigated. Considerable genetic diversity has been demonstrated using DNA fingerprints obtained by amplification with REP, ERIC, and BOX primers. Restriction analysis of the 16S-23S internal transcribed spacer (ITS1) allowed the strains to be subdivided into two major groups. In a phylogenetic tree, the ITS1s of these groups fell into two clusters, which also included the ITS1 of Pseudomonas syringae ("Syringae" cluster) and the ITS1 of P. fluorescens, P. tolaasii, P. reactans, P. gingeri, and P. agarici ("Fluorescens" cluster) from the GenBank database. Comparison of the ITS1 divergence levels within the "Fluorescens" cluster suggests expediency of treating P. tolaasii, P. reactans, various P. fluorescens groups, and, possibly, P. gingeri and P. agarici as subspecies of one genospecies. The intragenomic heterogeneity of ITS1s was observed in some of the pseudomonad strains studied. The results of amplification with specific primers and subsequent sequencing of the amplificate suggest the possibility of the presence of a functionally active syrB gene involved in syringomycin biosynthesis in the strains studied.     

Effects of Caenorhabditis elegans (Nematoda: Rhabditidae) on the spread of the bacterium Pseudomonas tolaasii in mushrooms (Agaricus bisporus)

The effects of mass-produced saprobic rhabditid nematodes, Caenorhabditis elegans on the spread of the bacterial blotch pathogen, Pseudomonas tolaasii , were studied in mushroom growth chambers. C. elegans significantly reduced the intensity of blotch on sporophores. Repeated isolations of the bacterial flora from the gut of C. elegans recovered from mushroom sporophores during cropping, revealed the presence of Pseudomonas fluorescens biovar reactans . All the isolates of P. fluorescens biovar reactans isolated from nematodes were antagonists of P. tolaasii .
C. elegans produced much larger populations in monoxenic cultures with P. fluorescens biovar reactans than with P. tolaasii . It is suggested that as C. elegans selects P. fluorescens biovar reactans rather than P. tolaasii as a food substrate it probably spreads the antagonist in the mushroom crop and may contribute to the control of bacterial blotch.     

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.     

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.     

Characteristics of pathogenic non-fluorescent (smooth) and non-pathogenic fluorescent (rough) forms of Pseudomonas tolaasii and Pseudomonas gingeri

Pseudomonas tolaasii and Ps. gingeri cultures isolated from naturally diseased mushrooms and cultures obtained from other workers were all observed to contain both smooth and rough colony forms. The smooth forms produced mucoid, non-fluorescent, glistening opaque colonies with entire margins. The rough forms produced non-mucoid, fluorescent, dull, translucent greenish-yellow colonies with irregular margins. Smooth forms were observed to produce a toxin and were pathogenic to mushrooms, whereas rough forms did not produce toxin and were non-pathogenic. Isolates of Ps. tolaasii were distinguishable from Ps. gingeri by various biochemical tests. In general, however, biochemical differences between the rough and smooth forms of each species could not be detected. Rough forms of Ps. tolaasii and Ps. gingeri remained stable in culture but smooth forms were unstable, tending to convert to rough forms at a very high rate.     

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.     

Pseudomonas tolaasii in mushroom (Agaricus bisporus) crops: activity of formulations of 2-bromo-2-nitropropane-1,3-diol (bronopol) against the bacterium and the use of this compound to control blotch disease

The bactericidal activity of 2-bromo-2-nitropropane-1,3-diol (bronopol) against Pseudomonas tolaasii , the causative organism of mushroom bacterial blotch, is enhanced by the addition of Tween 80, EDTA and phenylethanol. Results of tests with this pseudomonad confirm that bronopol is more active in alkaline solutions and enhancement of the bactericidal activity of this compound can be obtained by adding calcium carbonate, or mushroom casing (limestone and peat). Quantitative observations show that sterility can be achieved with bronopol at 100 µg/ml in 24 h following artificial inoculation of casing with Ps. tolaasii in glass flasks. On miniature mushroom beds in controlled environments a single application of bronopol, in tap water during routine watering, controls bacterial blotch disease. Bronopol is a slow-acting bactericide, destroying Ps. tolaasii in mushroom casing and effecting control of bacterial blotch disease.     

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.     

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.     

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.     

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.     

Multiple enzyme restriction fragment length polymorphism analysis for high resolution distinction of Pseudomonas (sensu stricto) 16S rRNA genes

Members of the genus Pseudomonas (sensu stricto) are important phytopathogens and agents of human infections, while other strains and species have beneficial bioremediation and biocontrol activities. Traditionally, these important species have been difficult to differentiate phenotypically; thus, rRNA lineage analyses have often been invoked. In this report, a newly developed approach is described to rapidly detect and distinguish fluorescent Pseudomonas isolates: PCR amplification of a Pseudomonas-specific 990-bp ribosomal RNA gene (rDNA) fragment [Appl. Environ. Microbiol. 64 (1998) 2545.] coupled with multiple enzyme restriction fragment length polymorphism (MERFLP) analysis using a single digestion mixture of AluI, HinfI, RsaI, and Tru9I incubated at 37 degrees C. The method distinguished 116 published sequences and 47 reference strains of authentic Pseudomonas representing 28 nomenspecies. A total of 55% (64/116) of the sequences analyzed by MERFLP were grouped into distinct phylogenetic clusters including Pseudomonas putida, P. syringae, P. aeruginosa, P. stutzeri, and P. fluorescens. The utility of the MERFLPs was confirmed when 100% (33/33) of the above named control reference strains were correctly placed into their phylogenetic clusters. The environmental relevance of the MERFLP method was confirmed when 67% of 28 forest and agricultural soil-derived presumptive Pseudomonas environmental clones and isolates were placed into the five major pseudomonad clusters, one clone fell into the P. agarici cluster, and five clones clustered near related pseudomonads. These data demonstrated that the PCR-MERFLP protocol provides an efficient and powerful tool for distinguishing isolates and rDNA gene libraries of environmental Pseudomonas species.     

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.