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.     

Influences of compost and casing layer depths on the mechanical properties of mushrooms

The mushroom growing bed consists of an underlying compost which is covered with casing (a peat and chalk or lime mixture) to induce fruit-body formation. The influences of the depths of the compost and casing layers on the mushroom texture, susceptibility to bruising, and ease of detachment from the growing bed surface were investigated. Textural properties were determined on cubes of cap tissue and detachment torque was determined by using an instrumented rotating suction cup which was lowered on to the caps. The proportion of mushrooms which detached at the base from the bed surface was recorded, since this is an important parameter for an automatic (robotic) harvesting system. Shallow casing resulted in mushrooms that were firmer and less likely to deform plastically, but that showed greater discolouration after a standard bruising treatment. Shallower casing also increased the proportion of mushrooms that detached at the base. Increasing the depth of compost produced firmer mushrooms, significantly increased the detachment torque and reduced the proportion of mushrooms detached at the base. The proportion of mushrooms that detached at the base correlated negatively with the plastic deformation of cap tissue. A positive correlation was found between mushroom dry matter content and tissue firmness.     

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.     

Physiologic response of Agaricus subrufescens using different casing materials and practices applied in the cultivation of Agaricus bisporus

Casing materials and practices used in the cultivation of Agaricus bisporus were evaluated in the cultivation of Agaricus subrufescens, using the best techniques for optimization of production, including the possibility of re-casing of the compost for the production of a second crop of mushroom. Casing based on peat moss, loam soil or coir was compared to casing material mixed with or without spawn-run compost. Based on the results, we conclude that the casing layer used in the cultivation of A. subrufescens should not necessarily be the same as that used in the cultivation of A. bisporus. For the tested strain cultivated with loam soil as casing layer, the ruffling technique is highly superior to CACing and should be pursued in further research. The re-casing of compost in new cycles showed good results suggesting that the currently used compost could be improved.     

The control of paedogenetic cecid larvae in mushroom beds by the use of thionazin

Thionazin incorporated in the casing layer of mushroom beds was shown to be highly phytotoxic even at 5 ppm, but when incorporated in compost it caused little phytotoxicity up to 20 ppm. A rate of 8 ppm gave satisfactory control of Heteropeza pygmaea Winnertz (Dipt.: Cecidomyiidae), but was less suitable against Mycophila speyeri (Barnes), for which the use of γ-BHC in the casing should be retained. A severe infestation of M. speyeri was shown to depress the total yield of mushrooms by 59% and the saleable yield by 87%.     

EXPERIMENTS WITH SOILS USED FOR CASING BEDS OF THE CULTIVATED MUSHROOM, PSALLIOTA CAMPESTRIS

In the present paper an account is given of observations and experiments on the influence of the casing soil on the cropping of beds of the cultivated mushroom, Psalliota campestris. It is shown from the results of a survey of cropping at thirty-nine mushroom farms that when the compost and cultural conditions are suitable for cropping, the production of mushrooms may be largely determined by the soil used for casing. Casing soils are classified into four grades depending on the numbers of sporophores formed, their rate of growth and the type of mushroom produced. A laboratory method of assessing the cropping potentialities of soils is described, and examples are given of its use for investigating field factors affecting the selection of soils for casing and the nature of the soil factors affecting cropping. It is suggested that chemical substances or specific surfaces in soils may promote cropping, but that close contact with air is necessary for them to be effective. It is concluded that direct trial is the only reliable method of assessing the value of a material for casing and that considerable fundamental research is needed on casing soils.     

Location of immature stages of the mushroom insect pest Megaselia halterata in mushroom-growing medium

Experiments were conducted to examine the location of oviposition by the phorid fly Megaselia halterata (Wood) (Diptera: Phoridae) in uncased and cased compost. Clearly, a majority of the gravid females choose oviposition sites directly after entering the top layer of the compost. In uncased compost, 60% of all adults emerged from the top of four compost layers of equal thickness. When the compost was covered by a casing layer which was still uncolonized by Agaricus bisporus, oviposition was further concentrated in the top compost layer. In this situation, 91% of all adults emerged from the top compost layer whereas only 1.5% emerged from the casing. When the casing layer was colonized by mushroom mycelium, 45% of all adults emerged from the casing layer and 53% emerged from the top compost layer. Further concentration in the top compost layer and the casing layer occurred as a result of upward migration of larvae. When compost was cased after oviposition, up to 43% of all adults emerged from the casing layer. We concluded that in the control of phorid infestations with insect pathogenic nematodes, applications in uncased compost can be restricted to the upper compost layer. When compost and casing are filled simultaneously, nematode applications in the casing layer only could be considered.     

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.     

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.     

Modelling the effect of the physical and chemical characteristics of the materials used as casing layers on the production parameters of Agaricus bisporus

The aim of this research was to show the mathematical data obtained through the correlations found between the physical and chemical characteristics of casing layers and the final mushrooms’ properties. For this purpose, 8 casing layers were used: soil, soil + peat moss, soil + black peat, soil + composted pine bark, soil + coconut fibre pith, soil + wood fibre, soil + composted vine shoots and, finally, the casing of La Rioja subjected to the ruffling practice. The conclusion that interplays in the fructification process with only the physical and chemical characteristics of casing are complicated was drawn. The mathematical data obtained in earliness could be explained in non-ruffled cultivation. The variability observed for the mushroom weight and the mushroom diameter variables could be explained in both ruffled and non-ruffled cultivations. Finally, the properties of the final quality of mushrooms were established by regression analysis.     

Effects of Caenorhabditis elegans (Nematoda: Rhabditidae) on yield and quality of the cultivated mushroom Agaricus bisporus

Thirteen species of saprobic rhabditid nematodes (11 genera) were identified from samples of compost and casing material collected from mushroom farms in the British Isles. Caenorhabditis elegans, the most frequently found saprobe, was mass-produced monoxenically and its effects on the cultivated mushroom, Agaricus bisporus (strain U3) were studied. C. elegans did not multiply in well-prepared, pasteurised, spawned compost, whereas casing material proved to be a highly suitable environment for its reproduction. An initial casing inoculum of 10⁶ nematodes/crate of compost (7.5 kg), caused a significant reduction in mushroom yield. Losses in total mushroom yields of 11%, 20% and 26% were caused by initial inoculum rates of 10⁶, 10⁷and 2 × 10⁷ nematodes/crate, respectively. Yields were negatively correlated with the initial nematode inoculation level and regression equations were derived. The nematode treatments caused fewer mushrooms to be produced and an absence of the usual distinctive flushing patterns. C. elegans caused considerable deterioration in mushroom quality and characteristic distortion of mushrooms. Individual sporophores were mis-shapen, notched and had brown or violet coloured grills. Up to 3.8%, 6.7% and 10.8% of total weight and 3.5%, 5.4% and 8% of total numbers of mushrooms were distorted at the three highest nematode inoculum rates tested. Weights and numbers of distorted mushrooms were positively correlated with the initial nematode population. C. elegans commonly colonised sporophores.     

Characteristics of a Hydrated, Alginate-Based Delivery System for Cultivation of the Button Mushroom

The production of the button mushroom Agaricus bisporus with mycelium-colonized alginate pellets as an inoculant of the growing medium was investigated. Pellets having an irregular surface and porous internal structure were prepared by complexing a mixture of 1% sodium alginate, 2 to 6% vermiculite, 2% hygramer, and various concentrations of Nutrisoy (soy protein) with calcium chloride. The porous structure allowed the pellets to be formed septically and then inoculated and colonized with the fungus following sterilization. By using an enzyme-linked immunosorbent assay (ELISA) to estimate fungal biomass, the matrix components of the pellet were found to be of no nutritive value to A. bisporus. Pellets amended with Nutrisoy at a concentration of 0.5 to 8% supported extensive mycelial growth, as determined by significantly increased ELISA values, with a concentration of 4% being optimal and higher concentrations proving inhibitory. The addition of hydrated, mycelium-invaded pellets to the compost or casing layer supported the thorough colonization of the growing substrate and culminated in the formation of mushrooms that showed normal development and typical morphology. Yields and sizes of mushrooms were comparable from composts seeded with either colonized pellets or cereal grain spawn. Similarly, amending the casing layer with pelletized-mycelium-colonized compost resulted in a 2- to 3-day-earlier and more-synchronous emergence of mushrooms than with untreated casing. This technology shows the greatest potential as a pathogen-free inoculant of the casing layer in the commercial cultivation of mushrooms.     

Comparative assessment of chelated spent mushroom substrates as casing material for the production of Agaricus bisporus

Spent mushroom substrate (SMS) leached with water or treated with chelating agents to remove metal cations, pasteurised to remove any harmful micro-organisms and mixed with peat has potential as a casing material for mushroom production. The microbial and chemical changes in SMS after treatment with citric acid, ethylene diaminetetraacetic acid (EDTA) and water were compared; treatment with the chelating agents resulted in lower ash content, conductivity and minerals, higher fibre fractions, carbon, hydrogen and nitrogen. The microbial and chemical changes in the materials after treatment with the two chelators and water were compared. Blending peat with the heat-treated materials at a ratio of 1:1 resulted in improved physical properties. The casings prepared from the test materials and the control, consisting of 100% peat, were compared after neutralising with lime for their productivity in a mushroom yield trial. As expected, the compost bags cased with the control were the most productive compared to the other casings. Of the three treated materials, casing prepared from SMS treated with EDTA blended with peat was the most productive. Dry matter of harvested mushrooms from chelated-SMS casings was significantly higher than the control casing. Comparison of the main components of peat and chelated SMS revealed that the major differences were in the proportions of ash, lipid, lignin and fibre fractions. The stability of some of these components, when complexed with metal cations present in lime may play an important role in determining the composition of the cell wall in fruiting bodies leading to high dry matter content. Received: 19 November 1998 / Received revision: 29 March 1999 / Accepted: 6 April 1999     

Effects of bendiocarb and diflubenzuron on mushroom cropping

When mixed into the casing or compost layers of a mushroom bed in the absence of pests, bendiocarb decreased yield and number of mushrooms according to concentration. The most severe effects were on mushroom number at the two highest rates used (100 and 1000 μg/g), and there were large increases in mushroom size. Effects of bendiocarb incorporation in the compost diminished with time, and there was partial compensation in yield and numbers at the fourth flush. The action of bendiocarb persisted when it was mixed into the casing. Diflubenzuron showed some opposite effects at lower concentrations. When either mixed into, or drenched onto the casing at the commercial rate (30 μg/g), yield and size were both increased and the timing of the flushes was unaffected. At the two higher concentrations (180 and 1080 μg/g), reductions in yield and number and an increase in mushroom size were shown. However, these effects became more severe with time, especially those on mushroom number, possibly due to the accumulation of a toxic breakdown product.     

Properties of peat-based casing soils and their influence on the water relations and growth of the mushroom (Agaricus bisporus)

Different combinations of peat and chalk or lime sources with differing moisture contents were used to determine how specific physical and chemical properties of the casing soil relate to the growth and water relations of the mushroom. The peat types varied in terms of decomposition and extraction method; the lime addition varied in terms of rate and type (chalk or sugar beet lime). During the colonisation of the casing soil before fruiting, the extension growth rate of mushroom mycelium was most closely correlated (negatively) with the volumetric moisture content of the casing soil. Scanning electron microscopy showed that mycelium growing at a lower casing soil matric potential (Ψ_m) had a much finer and branched structure than mycelium growing at a higher Ψ_m. Across all the peat and lime source treatments, a relationship was found between the mean Ψ_m of the casing soil and mushroom yield, with an optimum Ψ_m of -7.9 to -9.4 kPa. Mushrooms are produced in ‘flushes’ at about 8-day intervals and during the development of each flush of mushrooms, there was a significant decrease in casing soil Ψ_m . This decrease (to below -40 kPa) was greatest in the second flush, which was the highest yielding. There were no relationships between mushroom yield and casing soil osmotic potential Ψ_π within the range -93 to -154 kPa or any of the other chemical properties and water and air holding characteristics of the casing soils which were determined. Across different casing soil treatments, mushroom dry matter content was negatively correlated with mushroom yield and positively correlated with mushroom tissue osmotic potential. This revised version was published online in June 2006 with corrections to the Cover Date.     

Incidence,identification and pathogenicity of Cladobotryum mycophilum,causal agent of cobweb disease on Agaricus bisporus mushroom crops in Spain

Between 2008 and 2011, outbreaks of cobweb were observed in commercial white button mushroom (Agaricus bisporus) crops in Castilla‐La Mancha (Spain). In the last 3 years, the presence of the disease has notably increased resulting in serious economic losses. Based on morphological and genetic analyses, the casual agent of cobweb was identified as Cladobotryum mycophilum. A. bisporus mushroom crops were surveyed over a 2‐year period to estimate the incidence of cobweb. The presence of the disease was detected in 32% of the mushroom crops observed, being of particular concern in autumn (44% of crops infected) and winter (37%). As regards the casing material, the percentage of crops affected by cobweb was 34% in crops using mineral casing and 29% in those cased with a peat‐based casing, with no statistical relationship between the casing and the presence of cobweb. Two cropping trials inoculated with C. mycophilum were set up to evaluate the pathogenicity of the causal agent of cobweb in three peat‐based casings (C1, C2 and C3). The effect of cobweb on mushroom productivity was evaluated by comparing mushroom production and the cobweb patches detected in the casing soil. The decrease in total yield of mushrooms attributed to cobweb reached 12.9% with C2, and the crop area colonised by cobweb reached a final percentage of 36% with C3.     

The effect of spent mushroom compost on Lecanicillium fungicola in vivo and in vitro

Leached spent mushroom compost (SMC) and its extract were tested to suppress Lecanicillium fungicola in white button mushroom. Sterile and non-sterile mixture of SMC and peat were used to assess suppressiveness against L. fungicola in greenhouse experiments. The extract of SMC was prepared with sterile, non-sterile, filtered, supplied with nystatin, streptomycin and penicillin antibiotics to evaluate their effect in suppression of pathogen in vitro. Isolated bacteria from SMC extract were tested for antagonism rate against Lecanicillium fungicola. The results of the experiments showed that all applications rate of none-sterile SMC were effective in control of pathogen. However, the sterile SMC amendments did not have a positive effect on the pathogen suppression in vitro or in vivo, as was expected. The treatments amended with SMC 100% and 60% showed the most suppressive effect in the control of pathogen. Using of non-sterile SMC 20%, 40%, 60% and peat soil were most effective in mushroom yield. The extract of leached SMC showed inhibition of L. fungicola in petri dishes. Three bacteria isolated from extract, Bacillus subtilis, Bacillus licheniformis and Bacillus amyloliquefacien identified using 16s rRNA, showed an antagonistic effect with the fungal growth.     

Re-supplementing and re-casing mushroom (<Emphasis Type="Italic">Agaricus bisporus</Emphasis>) compost for a second crop

Experiments were performed to determine the effect of adding nutrient supplements to colonized mushroom compost (MC) for the production of a second crop of mushrooms. Mushrooms were harvested for 1, 2 or 3 flushes, the casing removed and the MC then was fragmented and re-supplemented with delayed release supplements treated or non-treated with fungicide (thiophanate-methyl; Topsin M 70WP) and re-cased. Overall double-crop yields were higher when MC was re-supplemented after 1st flush (1st flush MC) as compared to re-supplementation after the 2nd or 3rd flushes. Mean double-crop BEs were 128, 119 and 109% when 1st-, 2nd- and 3rd-flush MCs were used, respectively. Treatment of delayed release supplement with thiophanate-methyl fungicide did not affect mushroom yields. Soluble salts and potassium concentrations increased 350 and 900%, respectively, in the casing overlay through three flushes suggesting that removal of the casing would help to alleviate the build up of these potential growth-limiting materials. Re-supplementing and re-casing of MC represents a potential opportunity for growers to increase revenues and reduce costs associated with preparation and disposal of compost. The ability to double-crop mushroom compost would provide growers a chance to increase yields by 40% or more, depending on whether they re-supplement and re-case after 1st, 2nd or 3rd flush.