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 tolaasi in Mushroom Crops: A Note on Primary and Secondary Sources of the Bacterium on a Commercial Farm in England

Primary sources of Pseudomonas tolaasi Paine on a mushroom farm were the peat and limestone used in the casing process. The pathogen could not be detected in the farm soil, water supply, the mushroom spawn used, or in compost after spawning, but was isolated from the casing (peat/limestone mixture) layer of symptom-free mushroom beds and both the casing layer and compost of beds bearing diseased mushrooms. Secondary sources were numerous once the pathogen was present in mushroom beds. These included symptomless and diseased mushrooms, the fingers and shoes of people handling the crop, their baskets, knives and ladders. Ps. tolaasi could be isolated from dust in the air in infected houses and also from floors. Spores of infected mushrooms may transport the bacterium, as did sciarid flies and mites which are common pests of mushroom crops.     

WLIP, a lipodepsipeptide of Pseudomonas 'reactans', as inhibitor of the symptoms of the brown blotch disease of Agaricus bisporus

A cell-free crude extract containing the white line inducing principle (WLIP), a lipodepsipeptide produced by Pseudomonas 'reactans' , could inhibit browning of mushrooms caused by Pseudomonas tolaasii . Mushrooms inoculated with Ps. tolaasii at concentrations of 2·7 × 10⁶ cfu ml⁻¹ or higher showed the symptoms of the disease after 2 d of incubation. Mushroom caps treated with various concentrations of a crude WLIP preparation, and later inoculated with bacterial concentrations higher than the threshold value, did not develop the symptoms of the disease. One milligram of a crude WLIP preparation could block 50% of the symptoms caused by 1·2 × 10⁷ cfu. The inhibition of browning was effective when incubating at low temperatures for 4 d. A suspension containing 1·6 mg ml⁻¹ of pure WLIP was also able to inhibit the symptoms of brown blotch disease induced by 7·6 × 10⁶ cfu ml⁻¹ of Ps. tolaasii .     

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.     

An Ecological Study of Marked Rhizobium trifolii Strains on the Host Plant Trifolium repens var. Huai in an Acidic Peat and a Neutral Mineral Soil

An ecological study of the nodulation of Trifolium repens var. grassland Huai by genetically marked Rhizobium trifolii was carried out in two Irish soils, a neutral mineral and an acidic peat. An indigenous population of 2 x 10⁴ R. trifolii /g was found in the mineral soil. In the peat soil, 4 x 10¹ R. trifolii /g was found in the uninoculated peat. This number increased to 4.5 x 10⁵ R. trifolii /g, however, eight weeks after the peat soil was neutralized, supplemented with nutrients and sown with uninoculated clover seed. Indigenous R. trifolii strains from the mineral soil were effective whereas strains from the peat soil were ineffective on the host plant T. repens under plant room conditions. The introduced strains were inoculated on to clover seed at the rate of 1 x 10⁵ R. trifolii /seed. In the mineral soil, the introduced inoculum failed to establish at any period during the growing season. In the peat soil, the percentage establishment of the introduced inoculum varied from 40-50% of nodules selected eight weeks after sowing to 70-90% of nodules selected at the end of the growing season.     

Role of siderophores in the biocontrol of Pseudomonas tolaasii by fluorescent pseudomonad antagonists

A number of pseudomonad bacteria were investigated for their in vitro antagonism on agar against Pseudomonas tolaasii , the causative agent of bacterial blotch of the cultivated mushroom. Addition of FeCl₃ to the culture medium suppressed the antagonism in 14 of the 20 bacteria tested and the production of a substance with an absorption peak at 400 nm by all antagonists was eliminated by the presence of Fe³⁺. Both observations indicated that siderophores could be involved in the mode of action of some antagonists. The addition of the iron chelators EDTA and bipyridyl to the medium used for culture of antagonists and pathogens did not generally prevent growth. Siderophore production is not essential for the in vivo activity of antagonists.     

The bacterial population of a blanket peat

The number of aerobic bacteria in a blanket peat decreased with depth from 26 times 10⁶/g dry peat in the surface layers to 0.5 times 10⁶/g dry peat at 30–40 cm down the profile, thereafter remaining roughly constant. Obligate psychrophiles comprised <2.5% of this population. Anaerobes were most numerous, 9 times 10⁶/g dry peat at 6–10 cm depth, decreasing to 0.5 times 10⁶/g at 20–30 cm. Calculations indicated that these counts, 10³–10⁴-fold lower than the direct counts, substantially underestimated the active microbial population. Gram negative rods, the predominant aerobes in the surface layers, were replaced by unidentified Bacillus strains at 10–20 cm depth but became increasingly more numerous further down the profile. The Gram negative rods were the most numerous organisms/m² but the Bacillus strains, one third of which were present as spores, made the largest contribution to the biomass/m². Gram positive cocci, Arthrobacter and, infrequently, Nocardia were also isolated. Actinomyces -like forms were the predominant obligate anaerobes and were approximately three times more numerous than clostridia and a curved Gram negative rod.     

A Note on the Inhibition of Pseudomonas aeruginosa by a Modification of Brilliant Green Agar for Improved Salmonella Isolation

A strain of Pseudomonas aeruginosa having colonies that resemble those of salmonellas on brilliant green agar is almost totally inhibited by the addition of 1.0 mg/ml of sulphacetamide to the medium. Low numbers of Ps. aeruginosa grew equally well on brilliant green and nutrient agar, but 10⁶–10⁷ organisms were needed before any growth appeared on the medium containing sulphacetamide. During 12 months of routine use of the sulphacetamide medium, involving almost 3000 plates, Ps. aeruginosa has been isolated as a contaminant only once. Forty-seven salmonella serotypes were grown on the sulphacetamide brilliant green agar in the same period.     

Bacteriological stability and growth kinetics of Pseudomonas aeruginosa in bottled water

Bacteriological stability of water bottled in plastic containers and the growth kinetics of Pseudomonas aeruginosa were determined. Samples of water from the source, water to be bottled, finished product and sterile water bottled in non-returnable and returnable containers were analysed for aerobic colony count, coliforms, Escherichia coli and Ps. aeruginosa. The samples were examined for up to 30 d storage. Aerobic colony count increased 6 d after bottling to between 10³ and 10⁵ cfu ml⁻¹. Coliforms and E. coli were not found in any sample. Pseudomonas aeruginosa was isolated from commercial products bottled in returnable plastic containers due to the contamination from the containers and the subsequent multiplication utilizing trace nutrients. The predominant Ps. aeruginosa strains showed high doubling time (26 h) due to competition from the accompanying flora. In the absence of competing flora Ps. aeruginosa reached higher density than the maximum reached by aerobic flora, with a doubling time of only 3·6 h. After 30 d storage, this micro-organism was predominant.     

Heterotrophic bacterial populations in the mineral waters of thermal springs in Spain

The microbiological quality and heterotrophic bacterial populations of 26 thermal mineral water springs in Spain were studied. In most of the springs the number of viable aerobes was less than 10³ cfu ml^-1 and the number of sporulated bacteria less than 10² cfu ml^-1. No significant differences were foundin the counts obtained with Plate Count Agar (PCA) and PCA diluted 1 : 10 and incubated at 22°, 37° and 45°C. Total coliforms were found in 14 springs, faecal streptococci in three, spores of sulphite-reducing Clostridium and Pseudomonas aeruginosa in seven. Neither Escherichia coli nor Staphylococcus aureus were found. A total of 665 strains were isolated and 85·4% of these identified; 329 were Gram-positive and 239 were Gram-negative. The genera most prevalent present in the springs were Pseudomonas (in 92.3%), Bacillus (65.4%), Enterobacter, Micrococcus and Staphylococcus (50%), Acinetobacter (42.3%), Arthrobacter (38.4%), Clostridium (27%) and Xanthomonas (23%). Gram-negative bacteria predominated in the mesothermal springs and Gram-positive bacteria in the hyper- and hypothermal springs. The most common Gram-negative rod species isolated were Ps. fluorescens, Ps. aeruginosa, Ps. putida, Ent. agglomerans, Ent. sakazakii, Ac. calcoaceticus and Ent. amnigenus.     

Bioremediation of colour of methyl violet and phenol from a dye-industry waste effluent using Pseudomonas spp. isolated from factory soil

Methyl violet, a basic dye, is manufactured using phenol and dimethylaniline as the raw materials. It is, therefore likely that the waste effluent arising from such units may contain the dye and unused chemicals. Since such pollutants may be toxic, their removal becomes necessary. The studies were therefore aimed at their bioremediation using microbial species.
Four species of Pseudomonas , namely Ps. alcaligenes, Ps. mendocina, Ps. putida biovar B and Ps. stutzeri , were isolated from cattle dung enrichments and the soil samples in the premises of the factory manufacturing methyl violet, nearby Pune.
All the four species of Pseudomonas were able to remove phenol and methyl violet with simultaneous reduction in chemical oxygen demand (COD), total organic carbon (TOC) and ammoniacal nitrogen from the waste effluent and the removal was found to be optimum at the original alkaline pH (range 7.45–10.6) of the waste effluent, at ambient temperature (28 ± 2°C under aerated culture condition, at inoculum density of 1.5 × 10⁸ cells ml⁻¹ of the waste effluent and incubation period of 24–48 h. Thus, Pseudomonas spp. isolated from the environment of the dye industry itself and thus naturally adapted could be used for bioremediation of these pollutants.     

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.     

Survival of κ-carrageenan-encapsulated and unencapsulated Pseudomonas aeruginosa UG2Lr cells in forest soil monitored by polymerase chain reaction and spread plating

Abstract A method was developed for direct extraction, purification and amplification of DNA from forest soil. Eighty-two % of the DNA in Pseudomonas aeruginosa UG2Lr introduced into soil was recovered. The detection limit for the strain was approximately 800 cfu g⁻¹ of dry soil based on the polymerase chain reaction (PCR). Survival of κ-carrageenan-encapsulated and unencapsulated UG2Lr was monitored by antibiotic selective and bioluminescence-based nonselective plating and PCR-amplification of a tnsA fragment. After freeze-thaw treatment of soil samples, the unencapsulated UG2Lr declined from an initial population density of 1 × 10⁹ cfu g⁻¹ of dry soil to below the detection threshold of both selective (14 cfu g⁻¹ of dry soil) and nonselective (1 × 10³ cfu g⁻¹ of dry soil) plating. However, presence of nonculturable UG2Lr cells in the soil was revealed by PCR and resuscitation of the bacteria. Population density of the encapsulated UG2Lr increased from 2.7 × 10⁶ to 2.9 × 10⁸ cfu g⁻¹ of dry soil after a 3-week incubation at 22°C and declined to 6.3 × 10⁶ cfu g⁻¹ of dry soil after the freeze-thaw treatment.     

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.     

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.     

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.     

Molecular Detection of Phytophthora cryptogea on Calendula officinalis and Gerbera jamesonii Artificially Inoculated with Zoospores

In this work, a protocol for zoospores production of Phytophthora cryptogea , an economically important plant pathogen was optimized. Five different concentrations of zoospores (5 × 10⁵, 5 × 10⁴, 5 × 10³, 5 × 10², 5 × 10¹ zoospores/ml) from four different isolates of P. cryptogea (Maria 1, Maria 2, S3 1-A, Amazzone) were used as inoculum on pot marigold ( Calendula officinalis ) and gerbera ( Gerbera jamesonii ) plants. Maria 1 was the most virulent isolate both on pot marigold and gerbera plants according to disease severity. A rapid and sensitive pathogen DNA extraction protocol suitable for large quantities of plant samples was adopted. Conventional polymerase chain reaction (PCR) was able to detect the pathogen in artificially inoculated symptomless pot marigold (collected day 12) and gerbera plants (day 8) after pathogen inoculation, with the suspension of 5 × 10⁵, 5 × 10⁴, 5 × 10³ P. cryptogea  zoospores/ml. Real-time PCR showed the possibility to detect the pathogen in artificially inoculated symptomless pot marigold (collected day 8) and gerbera plants (day 4) after pathogen inoculation, with the suspension of 5 × 10⁵, 5 × 10⁴ P. cryptogea  zoospores/ml. The first symptoms appeared on pot marigold plants 14 days after pathogen inoculation and on gerbera plants 10 days after inoculation. Real-time PCR showed the possibility to detect the pathogen 4 days before conventional PCR and 6 days before the appearance of disease symptoms both on pot marigold and gerbera plants.     

Microbial populations in wetwood of European white fir (Abies alba Mill.)

Abstract A method for extraction of microbial populations from wood samples was worked out which gave good recovery of both aerobic and anaerobic microorganisms in agar shake dilution and plating enumerations. This method was applied to the quantification of microbial populations in three European white firs ( Abies alba Mill.) which were afflicted with the European fir disease. Low numbers of aerobic microorganisms (10²−10⁴ colony- forming units (cfu) per g fresh tissue) were detected in sapwood irrespective of the degree of affliction. Anaerobic bacteria were usually 1–2 orders of magnitude less frequent. Wetwood of highly diseased firs contained significantly higher numbers of aerobic microorganisms (10⁵−10⁷), whereas the number of anaerobes was not enhanced significantly. Among the prevalent aerobic microorganisms in wetwood were Protaminobacter, Pseudomonas strains, and a yeast. In anaerobic counts from wetwood, Klebsiella and Vibrio strains predominated. The sapwood contained Bacillus, Beijerinckia, Staphylococcus , and Clostridium spp. High numbers of aerobic microorganisms were also detected in the roots and lower stem of a diseased vine plant ( Vitis vinifera L.). The importance of microbial populations in wetwood formation and disease expression is discussed.     

Monitoring the fate of genetically engineered bacteria sprayed on the phylloplane of bush beans and grass

Abstract The fate of a Bacillus amyloliquefaciens with the recombinant plasmid pSB20 sprayed on the phyllosphere of grass, and of a Tn 5 marked Pseudomonas syringae sprayed on the phyllosphere of bush beans was studied in planted soil microcosms. B. amyloliquefaciens showed a decline from 1.5×10⁸ to 3.1×10² cfu g⁻¹ on the phylloplane of grass in the course of the experiment. B. amyloliquefaciens was easy to follow by selective cultivation due to the complete absence of bacterial background growth. Southern blot hybridization of Hin dIII digested genomic DNA showed plasmid restriction patterns identical with pSB20 indicating high plasmid stability. In total DNA extracts from phyllosphere bacteria the recombinant plasmid was detectable by Southern blot hybridization up to 6×10⁴ cfu g⁻¹ (wet weight). Counts of hybridizing colonies showed that P. syringae established on the phyllosphere of bush beans at between 5×10³ and 4×10⁶ cfu g⁻¹ fresh weight. During senescence of the bean plants the strain was no longer detectable by selective cultivation and subsequent colony hybridization. In contrast, Tn5 marked DNA was detected after PCR amplification over the whole period of the experiment.