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.     

Regulation of nitrogen-metabolizing enzymes in the commercial Mushroom Agaricus bisporus

Mycelium of Agaricus bisporus strain Horst U1 was grown in batch cultures on different concentrations of ammonium, glutamate, and glucose to test the effect of these substrates on the activities of NADP-dependent glutamate dehydrogenase (NADP-GDH, EC 1.4.1.4), NAD-dependent glutamate dehydrogenase (NAD-GDH, EC 1.4.1.2.), and glutamine synthetase (GS, EC 6.3.1.2.). When grown on ammonium, the activities of NADP-GDH and GS were repressed. NAD-GDH activity was about 10 times higher than the activities of NADP-GDH and GS. At concentrations below 8 mM ammonium, NADP-GDH and GS were slightly derepressed. When glutamate was used as the nitrogen source, activities of NADP-GDH and GS were derepressed; compared with growth on ammonium, the activities of these two enzymes were about 10 times higher. Activities of GDHs showed no variation at different glutamate concentrations. Activity of GS was slightly derepressed at low glutamate concentrations. Growth of A. bisporus on both ammonium and glutamate as nitrogen sources resulted in enzyme activities comparable to growth on ammonium alone. Activities of NADP-GDH, NAD-GDH, and GS were not influenced by the concentration of glucose in the medium. In mycelium starved for nitrogen, the activities of NADP-GDH, NAD-GDH, and GS were derepressed, while in carbon-starved mycelium the activity of GS and both GDHs was repressed.     

Lignin-Degrading Enzymes of the Commercial Button Mushroom, Agaricus bisporus

Agaricus bisporus, grown under standard composting conditions, was evaluated for its ability to produce lignin-degrading peroxidases, which have been shown to have an integral role in lignin degradation by wood-rotting fungi. The activity of manganese peroxidase was monitored throughout the production cycle of the fungus, from the time of colonization of the compost through the development of fruit bodies. Characterization of the enzyme was done with a crude compost extract. Manganese peroxidase was found to have a pI of 3.5 and a pH optimum of 5.4 to 5.5, with maximal activity during the initial stages of fruiting (pin stage). The activity declined considerably with fruit body maturation (first break). This apparent developmentally regulated pattern parallels that observed for laccase activity and for degradation of radiolabeled lignin and synthetic lignins by A. bisporus. Lignin peroxidase activity was not detected in the compost extracts. The correlation between the activities of manganese peroxidase and laccase and the degradation of lignin in A. bisporus suggests significant roles for these two enzymes in lignin degradation by this fungus.     

Genetic Diversity Analysis of Mycogone perniciosa Causing Wet Bubble Disease of Agaricus bisporus in China Using SRAP

In this study, 38 Mycogone perniciosa isolates of Agaricus bisporus from the main production areas in China were analysed for investigating the genetic diversity using sequence‐related amplification polymorphism (SRAP). A total of 132 polymorphic bands were obtained, ranging in size from 100 to 1700 base pairs. According to the dendrogram produced by the unweighted pair‐group method with arithmetic average of similarity coefficients from SRAP data, all the tested strains were divided into four clusters at a 71.6% similarity level. Strains 1–3 and 13–17 from, respectively, Xichong County of Sichuan Province and Yongchang County of Gansu Province were clustered in the same clade; strains 4–8 and 9–12 from, respectively, Long Hai City of Fujian Province and Luoyang City of Henan Province clustered in sa second clade; strains 18–21 from Wuhan City of Hubei Province grouped together in a third cluster distinct from the other strains.     

Uniparental Mitochondrial Transmission in the Cultivated Button Mushroom, Agaricus bisporus

A uniparental mitochondrial (mt) transmission pattern has been previously observed in laboratory matings of the cultivated mushroom Agaricus bisporus on petri dishes. In this study, four sets of specific matings were further examined by taking mycelial plugs from the confluent zone of mated homokaryons and inoculating these plugs into rye grain for laboratory fruiting and for fruiting under industrial conditions. Examination of the mt genotype of each individual fruit body for mt-specific restriction fragment length polymorphisms further confirmed that the mt genome was inherited uniparentally. The vegetative radial growth and the fruiting activity of two pairs of intraspecific heterokaryons, each pair carrying the same combination of nuclear genomes but different mt genotypes, were compared. Our results suggested that the mt genotype did not appreciably affect radial growth or fruiting activity. The failure to recover both heterokaryons, each carrying either parental mt genotype in any given cross, therefore clearly indicated that in matings of A. bisporus, the mt genome from one of the parental homokaryons is either selectively excluded in the newly formed heterokaryon or selectively eliminated in the immediate heterokaryotic mitotic progeny of the newly formed heterokaryon.     

Inhibitory Effects of Hexylresorcinol and Dodecylresorcinol on Mushroom (Agaricus bisporus) Tyrosinase

The effects of hexylresorcinol and dodecylresorcinol on the monophenolase and diphenolase activity of mushroom tyrosinase have been studied. The results show that hexylresorcinol and dodecylresorcinol can inhibit both monophenolase and diphenolase activity of the enzyme. The lag period of the enzyme was obviously lengthened, and the steady-state activity of the enzyme decreased sharply. Two microM of hexylresorcinol and dodecylresorcinol can lengthen the lag period from 98 s to 260 and 275 s, respectively. Both hexylresorcinol and dodecylresorcinol can lead to reversible inhibition of the enzyme. The IC50 values of hexylresorcinol and dodecylresorcinol were estimated as 1.24 and 1.15 microM for monophenolase and as 0.85 and 0.80 microM for diphenolase, respectively. A kinetic analysis shows that hexylresorcinol and dodecylresorcinol are competitive inhibitors. The apparent inhibition constant for hexylresorcinol and dodecylresorcinol binding with free enzyme has been determined to be 0.443 and 0.405 microM for diphenolase, respectively.     

Fruiting of Agaricus bisporus

Several enzymes were assayed in extracts from mycelium-colonised compost during growth and fruiting of Agaricus bisporus (Lange) Imbach. Comparison of changes of enzyme levels in axenic and nonaxenic cultures and in cultures of non-fruiting strains indicated that they were associated directly with the fungal mycelium. Large changes were found in the amounts of laccase and cellulase which were correlated with fruit body development. Laccase concentration increased during mycelial growth and then declined rapidly at the start of fruiting. Cellulase activity could be detected throughout growth but increased at fruiting. No such changes were observed in xylanase, alkaline protease, laminarinase and acid and alkaline phosphatases. Activities of laccase and cellulase were measured in axenic cultures arrested at various stages of fruiting development. Such cultures showed that the changes in concentration of laccase and cellulase were associated with the enlargement of fruit bodies.     

Mushroom (Agaricus bisporus) compost quality factors for predicting potential yield of fruiting bodies

A quality model has been developed from parameters determining the interactions of physical, chemical, and biological factors during the preparation of mushroom compost for growing Agaricus bisporus. Our results show that a partial least square model based on the combination of pH, dry matter, ammonia, carbon, hydrogen, ash, Cu, Fe, and Na could explain nearly 90% of the variation in mushroom yield obtained from four compost comparative trials. The yields in the data base for generating the model ranged from 138 to 305 kg per ton of compost. The validity of the yield model has been confirmed in a trial carried out in collaboration with experienced commercial growers. This has significant implications for compost producers, as production efficiencies can be maintained by targeting the important parameters.     

Influence of Steinernema feltiae and Diflubenzuron on Yield and Economics of the Cultivated Mushroom Agaricus bisporus in Dutch Mushroom Culture

The insect pathogenic nematode Steinernema feltiae (Nematoda: Steinernematidae) was shown to offer an alternative to the use of diflubenzuron for the control of the mushroom fly Lycoriella auripila (Diptera: Sciaridae). The influence of diflubenzuron and S. feltiae treatments on the yield and numbers of the mushroom, Agaricus bisporus , was studied. Applications of diflubenzuron after casing significantly reduced total mushroom yields by 10-13%. The greatest yield reductions were observed in the first flush. In the second flush, high yields were found despite significantly lower numbers of mushrooms. These high yields, however, could not compensate for the yield loss of the first flush. Some of the treatments with S. feltiae significantly increased yields, but these were not associated with a particular time of application. As nematodes did not reduce mushroom yields they were able to compete with diflubenzuron. Although the purchase costs of nematodes were high, their use was more economical than that of diflubenzuron when the yield losses of 10% due to casing treatments were taken into account.     

Inheritance of Strain Instability (Sectoring) in the Commercial Button Mushroom, Agaricus bisporus

The button mushroom, Agaricus bisporus, is a commercially important cultivated filamentous fungus. During the last decade, the button mushroom industry has depended mainly on two strains (or derivatives of these two strains). Using one of these highly successful strains (strain U1) we examined the phenomenon of strain instability, specifically, the production of irreversible sectors. Three “stromatal” and three “fluffy” sectors were compared with a healthy type U1 strain and with a wild-collected isolate. Compost colonization and fruit body morphology were examined. The main objective of this study, however, was to examine the meiotic stability of the sectored phenotype. Single basidiospores were isolated and subjected to a grain bioassay in which the ability to produce sectors was measured. Our results were as follows: (i) basidiospore cultures obtained from a wild-collected isolate showed no tendency to produce sectors; (ii) approximately 5% of the basidiospore cultures obtained from healthy type U1 strains produced irreversible sectors in the grain bioassay; (iii) the five primary sectors examined produced basidiospore cultures, half of which produced normal-looking growth in the grain bioassay and half of which produced some degree of sectoring; and (iv) the one sectored isolate that represented the F2 generation gave ratios similar to the 1:1 ratio observed for the F1 cultures.     

不同地区双孢蘑菇中蘑菇氨酸含量的比较研究

蘑菇氨酸曾被怀疑具有潜在的致癌效应,但其生理作用目前还存在争议.为比较分析我国不同地区双孢蘑菇中蘑菇氨酸含量,本研究采用反相高效液相色谱法检测了我国5个双孢蘑菇产区30份新鲜双孢蘑菇样品中蘑菇氨酸的含量.结果显示,我国双孢蘑菇中蘑菇氨酸含量范围较广,为155.6～934.4mg/kg FW.在此基础上,分析了菌株类型、培养料类型和采收季节等因素对蘑菇氨酸含量的影响.结果表明,不同菌株、不同栽培条件下生产的双孢蘑菇中蘑菇氨酸的含量差异明显.     

Optical and Potentiometric Study of the b- and c-Type Cytochromes in Mushroom Agaricus bisporus Lge Mitochondria

Differential spectrometry revealed two species for the b-type, as well as for the c-type, cytochromes in mitochondria from Agaricus bisporus Lge. The two b-type components are denoted according to their peak position in the α region at room temperature, i.e. b₅₆₀ and b₅₆₆. The b₅₅₆ component present in all the studied higher plant mitochondria was not detected in the system. At 293 K, the c-type cytochromes exhibit a common α band with a maximum at 550 nanometers. This band is split at 77 K, with peak positions at 547 nanometers (cytochrome c) and 552 nanometers (cytochrome c₁).     

Germination of basidiospores of Agaricus bisporus

The type of dormancy and conditions necessary for germination of Agaricus bisporus basidiospores (BS) were studied. BS failed to germinate on starvation agar and required the presence of carbon and nitrogen (asparagine and/or glucose) sources in the medium. Upon 3-week storage, BS germinated after 4-5 days. Heat shock (20 min at 45 degrees C) and the decreased temperature facilitated activation of germination. Heterocyclic compounds stimulating germination of endogenously dormant spores, such as furfural, failed to activate germination. The data obtained suggested an endogenous dormancy of A. bisporus BS differing from zygospores of Mucorales. BS contained 17-19% lipids with a composition of fatty acids differing from those of pileus and stipe of the fruiting body. The soluble carbohydrates of the cytosol amounted to 12% dry spore weight and consisted of mannitol (74%) and trehalose (26%). Unlike BS stored at 2 degrees C, the BS stored for 5 months at 20 degrees C lost their ability to germinate, which correlated with a decrease in the content of trehalose.     

Relative Numbers of Certain Microbial Groups Present in Compost Used for Mushroom (Agaricus bisporus) Propagation

The relative numbers of microorganisms associated with compost during mushroom production were studied by the dilution plate method. Thermophilic actinomycetes and fungi were isolated with a very high frequency early in the growing season. Although numbers of thermophilic bacteria diminished slowly during the season, the thermophilic fungi and actinomycetes diminished rapidly with the latter disappearing after 6 weeks. Mesophilic fungi other than Agaricus or Trichoderma remained relatively stable throughout the growing period. Agaricus could be isolated between the first and third break. Trichoderma became dominant after the fourth break. The mesophilic bacterial counts diminished during the most productive portion of the mushroom cropping season and then increased to much higher numbers toward the end of the season.     

NMR lipid profile of Agaricus bisporus

Lipids extracted from freeze dried and powdered cultivated Agaricus bisporus by the Bligh and Dwyer method, were subjected to 1D-proton and 2D-COSY NMR analysis. The diacylglycerophospholipids, mono-, di- and tri-glycerides, ether lipids, sphingolipids and steroidal lipids were studied qualitatively and quantitatively. Our findings suggested that (a) ethanolamines and cholines were the predominant diacylphospholipids, (b) sterols, mainly ergosterol, were present in relatively large quantities and (c) the phospholipid fatty acid composition consisted almost exclusively of linoleic acid. This type of detailed data on lipid composition was accurately and rapidly obtained in one step, without chemical modification of the sample. Additional information on four classes of lipid, including their fatty acid composition was obtained after separating the total lipid extract by NH₂-aminopropyl Certify II Bond Elut solid phase chromatography and analysing the NMR spectra of each class of lipids. The results demonstrated the potential of the method for the study of plant metabolism, development and taxonomy.     

Germination of Basidiospores of Agaricus bisporus

The type of dormancy and conditions necessary for germination of Agaricus bisporus basidiospores were studied. Basidiospores failed to germinate on starvation agar and required the presence of carbon and nitrogen sources (asparagine and/or glucose) in the medium. Upon 3-week storage, basidiospores germinated after 4–5 days. Heat shock (20 min at 45°C) and decreased temperature facilitated activation of germination. Heterocyclic compounds stimulating germination of endogenously dormant spores, such as furfural, failed to activate germination. The data obtained suggested an endogenous dormancy of A. bisporus basidiospores differing from zygospores of Mucorales. Basidiospores contained 17–19% lipids with a composition of fatty acids differing from those of the pileus and stipe of the fruiting body. The soluble carbohydrates of the cytosol amounted to 12% dry spore weight and consisted of mannitol (74%) and trehalose (26%). Unlike basidiospores stored at 2°C, basidiospores stored for 5 months at 20°C lost their ability to germinate, which correlated with a decrease in the content of trehalose.     

The lipids of Agaricus bisporus.

A comparison of the lipid composition of the vegetative and reproductive stages of Agaricus bisporus revealed no major qualitative differences, although quantitative divergence exist. The glycolipids consisted of acylglucoses, acylmannitol, acyltrehalose and a glucosyloxyfatty acid. Two of the acylglucoses corresponded to a tetra-acylglucose and to either a di- or a triacylglucose. The phospholipids were distinctive in that phosphatidylcholine could not be detected. Phosphatidylethanolamine and phosphatidylserine were the major phosphoglycerides. Examination of the neutral lipids revealed the expected array of acylglycerols, free and esterified sterols, and free fatty acids. A substantial amount (26 to 33%) of the fatty acids of the neutral lipids from both sporophore and mycelium were apparently of chain length greater than C18. Linoleic acid was a minor component of the total neutral-lipid fatty acids but comprised about one-half of the total free fatty acids.     

Crosses among Homokaryons from Commercial and Wild-Collected Strains of the Mushroom Agaricus brunnescens (= A. bisporus)

A new technique for the production of hybrid strains of the cultivated mushroom Agaricus brunnescens is described. Homokaryons were recovered from regenerated protoplasts obtained from several heterokaryotic strains. A total of 16 novel hybrids were produced in 63 attempted crosses between paired homokaryons. Recovery of both homokaryons and hybrids was verified by analysis of restriction fragment length polymorphisms. Three of four hybrids fruited in small-scale tests, further confirming that the isolates were true hybrids. Colony morphology alone was found to be a poor indicator of hybrid status. In two instances, three homokaryons crossed successfully in all combinations, suggesting that there are at least three alleles at the putative mating-type locus. Crosses between homokaryons from commercial and wild-collected isolates indicated that these strains belong to the same biological species.