The nature of the microbial stimulus affecting sporophore formation in Agaricus bisporus (Lange) Sing

An apparatus is described in which pure cultures of Agaricus bisporus were maintained on composted media in filtered atmospheres free from (a) noxious concentrations of carbon dioxide, and (b) contaminating microorganisms. When grown on compost alone, cultures of A. bisporus did not produce sporophores. Their formation was however stimulated by a covering layer of an unsterilized mixture of peat and chalk (=‘casing’ soil). Autoclaving or fumigating ‘casing’ with propylene oxide decreased populations of contaminating bacteria and prevented sporophore formation. Populations of micro-organisms isolated from unsterile ‘casing’ contained bacteria which when added to pure cultures of A. bisporus stimulated fruit-body formation. Numbers of these stimulators increased when cultured on a carbon-free liquid medium exposed to atmospheres with ethanol, ethyl acetate and acetone or containing the volatile metabolites of A. bisporus. The ability to utilize these volatile chemicals was exploited in a selective technique for isolating sporophore stimulators where aqueous suspensions of mixed bacterial populations were exposed to atmospheres of these materials for 5 days, before aliquots were added to agar media subsequently gelled. The stimulatory bacteria were identified as, or closely related to, Pseudomonas putida.     

Salt tolerance of Agaricus bisporus in relation to water stress and toxicity of sodium ions

Using NaCl or polyethylene glycol (PEG) solutions to progressively decrease the external osmotic potential of the peat casing of the growing medium used to culture the mushroom Agaricus bisporus resulted in proportionately decreased yields of sporophores. Over the range of -0.07 to -0.37 MPa, the extent of decrease in yield was similar with both types of osmoticum. However, with further decrease in external osmotic potential (from -0.37 to -0.62 MPa) there was a further proportional decrease in sporophore yield with PEG but a complete suppression of sporophore production with NaCl. Treatments with both NaCl and PEG decreased the concentrations of P, Mg, K, Fe and Mn, but not N and Cu, in sporophore dry matter. Treatment with NaCl solutions increased the concentrations of Na and CI ions in sporophore dry matter and decreased the concentration of Ca; PEG solutions had no effect. Ion toxicity associated with excessive accumulation of Na and C1 ions, or ionic imbalance associated with the concomittant decrease in Ca ions appear to be additional factors to osmotic stress in decreasing yield of sporophores when the growing medium becomes highly saline. The critical concentration of NaCl which caused 10% reduction in sporophore yield was 28 mM; A. bisporus is, therefore, moderately salt-sensitive.     

Protease activity inAgaricus bisporus during periodic fruiting (flushing) and sporophore development

Protease activity from sporophores and mycelium of the mushroomAgaricus bisporus was assayed during periodic cropping (flushing) and from sporophores during maturation. When the sporophores were harvested at the same developmental stages (pins or buttons) during cropping, proteolytic activity of the sporophores was found to oscillate with the same periodicity as the flushing cycle. For pin mushrooms (an early stage of development), peaks of activity occurred during the interflush periods, whereas for button mushrooms (a later stage of development) peak proteolytic activity coincided with the periods of maximum production. The proteolytic activity in the mycelium remained low and varied little with time. Of the tissues within the sporophore, gill tissue had a higher activity than the stipe or pileus. The changes in activity during sporophore development or maturation depended on the period in the flushing cycle when the sporophore was initiated. The results are discussed in relation to the possible role and regulation of flush co-ordinated proteases.     

TRANSPIRATION FROM THE SPOROPHORE OF AGARICUS BISPORUS ‘WHITE’

The transpiration from normal, intact, growing sporophores of the cultivated mushroom, Agaricus bisporus cv ‘White’ was determined by a gravimetric method. A simple method was devised to estimate the surface area of a sporophore. Under different conditions of temperature and relative humidity, the quotient of transpiration/cm² sporophore surface area and evaporation/cm² free-water surface area did not significantly differ from 1. Transpiration from the underside of an open-veil mushroom was related to the planar area rather than to the total exposed gill area. Normally growing sporophores transpired up to 3 mg/cm²/hr. It was estimated that during development to the open-veil stage, a sporophore transpired a quantity of water equal to ca. one-half of its fresh weight. There was no evidence of factors other than environmental affecting the evaporation of water from the surface of the normally growing sporophore. Our data were not extensive enough, however, to provide evidence for or against Schütte's hypothesis that transpiration in a mature agaric fructification may be intimately linked with a physiological process.     

Lipid profile of Pleurotus sajor caju

The lipid profile of Pleurotus sajor caju was studied in relation to mycelial and sporophore growth and different cultural factors. The growth was characterised by lipid synthesis during mycelial growth and utilisation during sporophore growth. The degree of instauration increased during mycelial growth and decreased during sporophore formation. The fatty acid composition of mycelium and sporophore was similar, linoleic acid (C_18:2) being the most dominant acid in both. C:N ratio had a significant (P<0.05) positive effect on mycelial dry weight; however, per cent total lipids was similar. Non-polar lipids became more unsaturated as the temperature was raised from 10° to 25°C and pH from 3.0 to 6.0, but declined when the cultures were aerated. Mycelial dry weight increased significantly (P<0.05) when the liquid medium was supplemented with lipids. In general, fatty acids with carbon chain length C₁₆ and C₁₈ stimulated the growth of mycelium. Supplementation of solid substrate (cotton seed hulls) with safflower oil, soybean oil or rice bran significantly (P<0.05) increased the yield of sporophores. Total lipids and ratio of non-polar to polar lipids were not affected by lipid supplementation.     

Gibberellin-like Substances in the Sporophores of Agaricus bisporus (Lange) Imbach

Sporophores of cultivated Agaricus bisporus (Lange) Imbach,were shown to contain a gibberellin-like substance active inthe dwarf maize (d-5), -amylase and other bioassays. Ethyl-acetateextraction followed by paper, column, and thin-layer chromatographyrevealed the presence of one major active substance. Ficin hydrolysisof dried sporophore powder, after the complete removal of freesubstances, released more gibberellin-like substances, one ofwhich appeared identical to the free compound. The free substance was predominantly in the lamellae and residualpileus tissue. The major active substance released by ficinoccurred mostly in the lamellae but also in substantial equalamounts in both stipes and pilei. No activity was found in extractsof dikaryotic vegetative mycelium on malt agar. The level ofactivity in extracts from sporophores stored at – 20 °Cfell sharply after 7 d, and then remained constant over a periodof 6 weeks. The content of gibberellin-like substances in youngand old whole sporophores showed wide variation between experiments.In most cases young 2-d tissue had higher levels than old, 11-dtissue on a fresh-weight basis. Purified sporophore extractsand authentic gibberellins had no stimulating effect on growthof sporophores or of cultured vegetative mycelium. The inhibitorsof diterpene biosynthesis, CCC, and AMO-1618 induced a smallincrease in mycelial growth rate. Ethyl-acetate extraction ofhorse-straw compost prior to inoculation with Agaricus bisporusshowed the presence of gibberellin-like activity in significantamounts.     

Pigmented Bacteriostatic Substances and Amino Acids Produced by Phlebopus sulphureus and Phlebopus lignicola

The pigmented bacteriostatic substances and ninhydrin-reactive compounds produced in culture by sporophores, stipes, and residual medium of Phlebopus sulphureus and P. lignicola were studied chromatographically. Acetone extracts of both species demonstrated bacteriostatic activity against Bacillus subtilis and Escherichia coli, the maximal inhibition zone being 13 mm. The chloroform extract was inactive. Six compounds, mainly yellow, extracted in ethyl alcohol and separated by chromatography, were common to the sporophore of P. sulphureus and the stipes of P. lignicola. Eight amino acids, mainly sulfur-containing, were identified chiefly from the mature sporophore of P. sulphureus.     

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.     

A simple chemically,defined medium for the growth of Aphanomyces euteiches and some other oomycetes

A chemically-defined medium composed of glutathione, D-glucose, DL-asparagine, calcium and magnesium chlorides, and monobasic and dibasic potassium phosphates supported growth of several species of filamentous fungi which include seven isolates ofAphanomyces euteiches and single isolates ofA. leavis, A. stellatus, Achlya ambisexualis and several species ofPythium. Some growth occurred if a stoichiometric equivalent of the amino acids contained in glutathione were substituted for it. Dithiothreitol, a compound which keeps glutathione in the reduced form, inhibited growth ofA. euteiches at the concentrations tested. Replacement of the medium with a solution of known ionic composition caused the fungal colonies to produce and release zoospores and to produce oospores.     

II. Evidence of the presence in yeast extract of substances which stimulate the growth of Histoplasma capsulatum and blastomyces dermatitidis similarly to that found in starling manure extract

Summary A medium consisting of agar plus yeast extract contained the necessary metabolites for rapid growth and sporulation ofHistoplasma capsulatum andBlastomyces dermatitidis. H. capsulatum when harvested after 10 or 30 days incubation period from this medium was shown to have a similar number of spores as well as total particle viability for each period of growth.The growth characteristics ofH. capsulatum and four different isolates ofB. dermatitidis on yeast extract medium were similar to that obtained previously using starling (Sturnis vulgaris) manure extract medium. These characteristics are rapid growth consisting of many viable spores and a low ratio of vegetative mycelium.Several isolations ofH. capsulatum from naturally contaminated soil specimens were made using yeast extract medium.From the Communicable Disease Center, Public Health Service, U. S. Department of Health, Education, and Welfare.     

Formation of Biotinyl-CoA Synthetase,the First Enzyme Involved in Microbial Biotin Degradation

Various bacteria which can grow on biotin as a sole carbon source were isolated from soil samples. These bacteria were classified into three groups according to the biotin-degrading pattern. Cell-free extracts from all these bacteria grown on biotin possessed high biotinyl-CoA synthetase activities. Cultural conditions for strain No. 166, the highest biotinyl-CoA synthetase producer, were examined. Biotinyl-CoA synthetase was induced in the presence of biotin, but not in the presence of any other carboxylic acids or biotin intermediates. The addition of lactose and yeast extract to the medium enhanced both the growth and the enzyme activity. The enzyme reaction for biotinyl-CoA synthesis required ATP, CoA and Mg²⁺ as well as biotin. From taxonomical studies, bacterium No. 166 was identified as a strain of Mycoplana.     

Volatile compounds from the mycelium of the mushroom Agaricus bisporus

The pattern of volatiles from the mycelium of two commercial strains of Agaricus bisporus, grown in axenic culture on a semi-synthetic medium, was found to be broadly similar to that of the volatiles identified from sporophores. Tetrachloro-1,4-dimethoxybenzene, a known secondary metabolite of several Basidiomycetes, was found in the mycelium though not in the sporophores. [³⁶Cl]Tetrachloro-1,4-dimethoxybenzene was obtained when sodium [¹³Cl]chloride was added to the medium.     

Molecular and physiological diversity among Verticillium fungicola var. fungicola

The genetic and physiological variability of Verticillium fungicola var. aleophilum responsible for Agaricus bisporus dry bubble disease in North America is well documented but little is known about the var. fungicola affecting European crops. Variability was assessed within this variety and compared with that reported for the var. aleophilum. Eighteen isolates of V. fungicola var. fungicola and four var. aleophilum isolates were analysed for DNA polymorphism, mycelial growth, response to biochemicals produced by A. bisporus, fungicide resistance, and pathogenicity assessed by direct inoculation on sporophore or casing contamination. RAPD and AFLP markers delineated three French isolates from a homogeneous group containing the other var. fungicola isolates, but no correlation could be drawn between DNA polymorphism and the various traits studied. The var. fungicola isolates were more susceptible than the var. aleophilum isolates to the antibiosis effect of A. bisporus. Only mycelial growth rate at 23 °C could explain the variability in aggressiveness among the European isolates. The putative effect of the post-incubation temperature on contamination during mushroom cultivation was discussed. This work emphasized that, like the American var. aleophilum, the var. fungicola in Europe is genetically homogeneous, but physiological diversity exists, especially in France where it could be related to less standardized cultural practices.     

Competition between soil bacteria and fusarium

Summary The phenomenon of competition has been characterized in liquid medium and sterile soil systems using a variety of soil bacteria andFusarium oxysporum f.cubense as test organisms. For most of the bacteria, suppression of the fungus was the result of a biologically induced nitrogen deficiency, this effect being reversed by the addition of excess inorganic nitrogen. High populations of competitors were found in two soils of neutral pH, but no isolates competed in the acid San Alejo loam.Agrobacterium radiobacter was able to compete when San Alejo loam was limed to about pH 6.6. Inhibition of the fungus by a number of gram-positive, spore-forming rods could not be accounted for in terms of competition for nutrients or by antibiotic production in artificial media.The competitive ability ofA. radiobacter when tested in twelve Central American soils was found to be related to pH in acid and neutral environments but was correlated with texture, organic-matter content and total nitrogen in soils of intermediate pH. In all soils where inhibition occurred, the competitive effect was overcome by additions of inorganic nitrogen. Excluding the group ofBacillus spp., the competitive ability of soil bacteria was related to the ability to develop in the absence of amino acids and growth factors but could not be correlated with growth rates of the bacteria in soil or liquid medium.It is suggested that competition for nutrients is a significant means of ecological control among members of the soil microflora and, together with competitive interactions for space and oxygen, may be the major factors governing the biological control of soil-borne fungi.The investigation was supported in part by a grant from the United Fruit Company. Agronomy Paper No. 471.     

Untersuchungen zur Stimulation der Fruchtkörperbildung bei einemPleurotus aus Florida

Zusammenfassung Extrakte aus Fruchtkörpern vonPleurotus oderAgaricus fördern die Fruktifikation vonPleurotus-Mycel. Das äußert sich in der regelmäßigen Primordienbildung 7–10 Tage nachdem die Extrakte auf das Mycel gegeben worden sind und in erhöhten Fruchtkörper-(=FK-) Gewichten.Nach Fraktionierung der Extrakte durch Ultraoder Gelfiltration wurde eine starke Förderung der FK-Gewichte nur noch durch die Fraktionen mit niedrigeren Molekulargewichten festgestellt. Die Zahl der Anlagen wurde dagegen auch durch die hochmolekularen Bestandteile erhöht, aber nicht durch Protein alleine.Mit zunehmender Verdünnung der Extrakte nahmen die FK-Gewichte schneller ab als die Anlagenzahlen.40 mg L-Asparagin oder die äquimolare Menge Harnstoff wirkten ähnlich wie Extrakt aus 1 g Fruchtkörper. Zucker hatten keinen Effekt.Primordienbildung und FK-Wachstum dürften beiPleurotus zwei verschiedene Prozesse sein, wie beiAgaricus bisporus.Methoden zum Nachweis zweier hypothetischer Wirkstoffe werden diskutiert.

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Investigations on the stimulation of fruit body formation in aPleurotis from Florida

Summary Extracts from fruit bodies ofPleurolus orAgaricus promoted fructification ofPleurotus mycelium. This was visible in regular primordia formation 7–10 days after application of extract to the mycelium and in higher sporophore weights.After fractionating the extracts by ultra- and gel-filtration a marked stimulation of sporophore weights was detectable only in fractions of lower molecular weight. The number of primordia, however, was also increased by compounds of high molecular weight, but not by protein alone.With increasing dilution of the extracts the weight of fruit bodies decreased more rapidly than the number of primordia.40 mg of L-asparagine or equimolar amounts of urea showed an effect similar to that of the extract from 1 g fruit body. Suggars gave no reaction.Sporophore initiation and fruit body growth are supposed to be two different processes inPleurotus as well as inAgaricus bisporus.Methods for detecting a hypothetical sporophore inducer and an inhibitor are discussed.

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Herrn Prof. Dr.Reinhold von Sengbusch zum 70. Geburtstag in Dankbarkeit und Verehrung gewidmet.     

Observations on the bacterial oxidation of chlorophenoxyacetic acids

Summary Three bacterial strains, one ofF. peregrinum (Stapp and Spicher) and two Achromobacter strains, have been isolated from soil and shown to decompose either 2,4-D, MCPA orp-chlorophenoxyacetic acid. Aerobic conditions are essential for the bacterial decomposition of 2,4-D. Pretreatment of soil with one of the three chlorophenoxyacetic acids accelerated the rate of breakdown of either of the other two. In a liquid medium, growth of theF. peregrinum strain caused breakdown of 2,4-D and liberated 76% of the chlorine in 2,4-D in ionic form. An unknown acidic substance, colourless in acid solution but forming a yellow sodium salt has been detected in cultures ofF. peregrinum or an MCPA-decomposing Achromobacter strain growing inp-chlorophenoxyacetate medium. The bacterial oxidation of chlorophenoxyacetic acid herbicides was attributed to adaptive enzyme formation. Respiration experiments showed that the oxidation of 2,4-D or ofp-chlorophenoxyacetic acid is incomplete. 4-Chloro-2-hydroxyphenoxyacetic acid and 4-chlorocatechol may be metabolic intermediates in the case ofp-chlorophenoxyacetic acid, but no intermediary metabolites have as yet been established for 2,4-D.     

Effect of 1-aminocyclopropane-1-carboxylic acid deaminase producing bacteria on the hyphal growth and primordium initiation of Agaricus bisporus

The mechanism of casing soil stimulating the primordium formation of Agaricus bisporus is not well understood so far. Our results showed that 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase (AcdS)-producing bacteria were abundant in the casing soil of A. bisporus and accounted for up to 20 % of total culturable bacteria. A. bisporus produced ACC and ethylene. The supplement of methionine increased the ACC concentrations within the hyphae, and aminooxyacetic acid displayed an opposite effect. Methionine and ACC promoted the ethylene production while CoCl₂ suppressed the production. The AcdS-producing bacterial strain Pseudomonas putida UW4 co-cultured with A. bisporus could attach to hyphae, stimulate the hyphal growth, and reduce the ethylene production of A. bisporus. Added in sterilized casing soil, it induced the primordium formation of A. bisporus. In comparison, its AcdS-deficient mutant UW4-AcdS^? displayed the opposite effects. These results indicated that the inhibitor to the primordium formation of A. bisporus was ethylene; the AcdS-producing bacteria within the casing layer cleaved ACC, lowered the ethylene level in mushroom hyphae, and relieved the inhibition of ethylene. This is a new model of the synergism between bacteria and fungi.     

Formation of biologically active substances by rhizosphere bacteria and their effect on plant growth

Nine out of seventeen strains of bacteria with a pronounced effect on seed germination and on seedling growth, isolated from root surfaces and rhizosphere soil of maize, were selected for a study on the formation of biologically active substances. β-Indole acetic acid (45–72 μg/1.000 ml) was produced by four strains, gibberelline-like substances (1.0–60.0 μg/1.000ml) by all strains, biotin and pantothenic acid by the majority of strains and nicotinic acid by five strains. Amino acids were formed by all strains but in low amounts. Four strains produced growth inhibitors. The highest amounts of biologically active substances were found in cultures ofPseudomonas fluorescens andBacillus brevis. The various cultures ofPseudomonas fluorescens differed in their capability to produce biologically active substances. The majority of bacterial cultures or their supernatants significantly stimulated the germination of seeds and some of them significantly affected the growth of plants. Inoculation of maize seeds with strainsPseudomonas fluorescens andChromobacterium violaceum significantly increased the yield of dry matter of plants.