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.     

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.     

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.     

Bacteria Antagonistic to Pseudomonas tolaasii and their Control of Brown Blotch of the Cultivated Mushroom Agaricus bisporus

S ummary : Pseudomonas tolaasii was isolated from casing peat of healthy and diseased mushroom beds, compost of diseased mushroom beds and from soils round a mushroom farm. It was not isolated from fresh peat or compost from healthy mushroom beds. Three bacteria antagonistic to Ps. tolaasii were isolated from soil and peat. These were a nonfluorescent Pseudomonas sp. (closest to Ps. multivorans ) from soil; and strains of Ps. fluorescens and Enterobacter aerogenes from peat. When the antagonists and the pathogen were added in the ratio of 8 × 10⁷: 10⁶ cells/ml to unsterilized peat and applied to mushroom trays, infection of mushroom sporophores by the pathogen was effectively controlled. In vitro studies failed to show lysis or growth inhibition of Ps. tolaasii by the antagonists.     

Effects of bacteria isolated from a saprophagous rhabditid nematode Caenorhabditis elegans on the mycelial growth of Agaricus bisporus

P. S. GREWAL AND P. HAND. 1992. The effects of 10 species of bacteria isolated from a saprophagous rhabditid nematode Caenorhabditis elegans on mycelial growth of the cultivated mushroom Agaricus bisporus were studied in agar cultures. Bacterial species showed differential effects on the mycelial growth of A. bisporus and the effects also depended upon the mushroom strain (C43, C54 and U3). Bacillus cereus, Bacillus sp. and Enterobacter amnigenus caused significant inhibition in mycelial growth of all three strains of A. bisporus. Pseudomonas aeruginosa, Ps. fluorescens biovar reactans and Ps. maltophilia resulted in a significant increase in mycelial growth of C54 strain. Enterobacter cloacae caused a mean inhibition of about 83% in the linear mycelial extension of the most commonly cultivated mushroom strain U3. Bacillus cereus, Ent. amnigenus and Ent. cloacae produced volatile inhibitory substance(s). This is believed to be the first report about the inhibitory effects of specific bacteria isolated from a saprophagous nematode on the mycelial growth of A. bisporus.     

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.     

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 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.     

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.     

Toxicity of 2,4-diacetylphloroglucinol (DAPG) to Plant-parasitic and Bacterial-feeding Nematodes

The antibiotic 2,4-diacetylphloroglucinol (DAPG) is produced by some isolates of the beneficial bacterium Pseudomonas fluorescens. DAPG is toxic to many organisms, and crop yield increases have been reported after application of DAPG-producing P. fluorescens. This study was conducted to determine whether DAPG is toxic to selected nematodes. The plant-parasitic nematodes Heterodera glycines, Meloidogyne incognita, Pratylenchus scribneri and Xiphinema americanum, and the bacterial-feeding nematodes Caenorhabditis elegans, Pristionchus pacificus, and Rhabditis rainai, were immersed in concentrations ranging from 0 to 100 μg/ml DAPG. Egg hatch and viability of juveniles and adults were determined. DAPG was toxic to X. americanum adults, with an LD(50) of 8.3 μg/ml DAPG. DAPG decreased M. incognita egg hatch, but stimulated C. elegans hatch during the first hours of incubation. Viability of M. incognita J2 and of C. elegans J1 and adults was not affected. There were no observed effects on the other nematodes. The study indicated that DAPG is not toxic to all nematodes, and did not affect the tested species of beneficial bacterial-feeding nematodes. Augmentation of DAPG-producing P. fluorescens populations for nematode biocontrol could be targeted to specific nematode species known to be affected by this compound and by other antibiotics produced by the bacteria, or these bacteria could be used for other possible effects, such as induced plant resistance.     

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.     

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.     

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.     

Relative contribution of nematodes (Caenorhabditis elegans) and bacteria towards the disruption of flushing patterns and losses in yield and quality of mushrooms (Agaricus bisporus)

Laboratory tests of bacteria isolated from the body surface, or from the gut, of a saprophagous rhabditid nematode Caenorhabditis elegans infesting mushrooms (Agaricus bisporus) showed that some bacteria enhanced nematode reproduction and that others inhibited it. As some bacteria were shown to inhibit mycelial growth of the mushroom, the effects of Acinetobacter calcoaceticus var. anitratus, Enterobacter cloacae and Serratia liquefaciens, either alone or in combination with C. elegans, on the flushing patterns, quality and yield of A. bisporus (strain Horst U3) were studied. Bacteria alone had little effect on flushing patterns whereas C. elegans delayed the onset of mushroom production and significantly disrupted the growth pattern of crops, with mushrooms appearing more regularly and not within obvious flushes. Inoculation with bacteria resulted in ‘browning’ of mushrooms that was even more pronounced in C. elegans treatments. Characteristic distortion of sporophores was observed only in the presence of C. elegans. Nematodes commonly colonised sporophores. Bacteria affected the size of nematode populations both on the sporophores and in the casing. Significant yield loss occurred; up to 10% when bacteria were inoculated, up to 27.8% when C. elegans was inoculated, and up to 35% with both bacteria and nematodes. Synergism between C. elegans and A. calcoaceticus var. anitratus was observed; the combination resulted in significantly greater reduction in mushroom yield than any other treatment. It is concluded that bacteria contribute to yield loss and quality deterioration in A. bisporus but that the effects are far greater in the presence of C. elegans.     

Biological characterization of white line-inducing principle (WLIP) produced by Pseudomonas reactans NCPPB1311

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

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.