Sporophore production ofAgaricus bisporus in aseptic environments

Production of mature sporophores ofAgaricus bisporus was achieved for the first time in amended, autoclaved soil, gamma-sterilized soil, and soil-extract agar medium. The initiation of sporophores was triggered by metabolites of soil-inhabiting bacteria, particularly nodule forming isolates. Whether a single metabolite or several metabolites of these bacteria caused formation of sporophores could not be established; however, biotin alone when added to soil extract medium produced comparable results. The potentiality of different bacteria to induce sporophore formation varied considerably within species and isolates.Amino acids favored vegetative growth ofA. bisporus, but failed to induce formation of sporophores. Organic acids supported luxuriant growth and poor sporophore formation. Among several growth-promoting substances and vitamins, biotin induced abundant formation of mature sporophores.The authors are thankful to Dr. C. Corke, Department of Soil Microbiology, University of Guelph, Ontario, for providing some bacterial cultures used in this study.     

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.     

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.     

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.     

The Stimulation of Spore Germination in Agaricus bisporus by Living Mycelium

The stimulation of the germination of Agaricus bisporus sporesby mycelium of the same species has been shown to be due toa volatile metabolite, diffusing into the culture medium andinto the atmosphere. A wide range of other fungi has been foundto affect A. bisporus spores in a similar manner. There wasno evidence . that the stimulant was carbon dioxide. A seven-carbonolefin, isolated from air, which had been passed through culturesof A. bisporus mycelium and dried with phosphorus pentoxide,showed germination-stimulating activity but may have been producedby the action of this drying agent on some other metabolitefrom the mycelium. Volatile materials reported by other workersfrom A. bisporus mycelium and from Saccharomyces cereviseaewere tested and, of these, iso-valeric acid and iso-amyl alcoholwere found to stimulate spore germination of A. bisporus     

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.     

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.     

Photosporogenesis of Coprinus congregatus: Correlations between the physiological age of lamellae and the development of their potential for renewed fruiting

When the hymenial lamellae of Coprinus congregatus Bull ex Fr. are used as implants, their potential for renewed fruiting varies according to the photocontrolled meiosis and the consecutive sporogenesis. In the case of young lamellae, whose basidia are still at the dikaryon stage, one can observe immediate start of mycelial growth all around the lamellae and production of the first mature sporophores directly on the lamellae (direct fruiting). Simultaneously, meiosis does not occur in hymenial cells. Conversely, in the case of implantation of the oldest lamellae, whose basidia are characterized by meiotic nuclei beyond prophase 1 and rather near telophase 2 (tetranucleate stage), vegetative growth starts slowly and the first mature sporophores are not produced on the lamellae but on the surrounding vegetative mycelium (indirect fruiting). When the lamellae are isolated from photoindifferent primordia – for instance, 12 h before maturity – sporogenesis in hymenial cells proceeds normally until autolysis of the isolated lamellae. Such isolated lamellae no longer show direct fruiting where the first flush is concerned.     

Studies on two Gibberellin-like Substances in Young Shoots of Tomato (Lycopersicon esculentum Mill.)

Two gibberellin-like substances were found in the acidic fractionof shoot extracts of the tomato (Lycopersicon esculentum Mill.,cultivar Potentate). These were resolved by paper chromotographywith iso-propanol/ammonia/water (10:1:1) as the developing solventbut not with n-butanol/1.5 N ammonia (3:1). Both substanceswere active in the dwarf maize bioassay on mutants d-1, d-2,d-3, and d-5, and appeared to be more active on d-5 than d-1.Neither was active in the Meteor Pea assay. Neutral and basicfractions were inactive. The relative amounts of these two substances varied accordingto the age of the tissues from which they were extracted andthis feature is discussed in relation to future studies on thephysiology of gibberellin-like substances in vivo.     

Isolation of high quality and yield of RNA from Agaricus bisporus with a simple, inexpensive and reliable method

A method for isolating high purity and quantity RNA from Agaricus bisporus which is rich in proteins, carbohydrate, fiber and secondary metabolites, is described. RNA was extracted from mycelium, primordia, sporophores at two development stages and two post-harvest storage stages as well as from pileipellis, inner cap, gill and stipe of the mature sporophore. The A(260)/A(230) and A(260)/A(280) ratios of isolated RNA from fruiting bodies were both ~2 and the yield was about 200 μg/g fresh wt (FW). The yield of RNA from mycelium was approx. 100 μg/g FW. High quality RNA was also extracted from fruiting body tissues of Lentinus edodes, Pleurotus ostreatus, Flammulina velutipes and Pleurotus eryngii with yields from 130 to 225 μg/g FW. RNA extracted from all samples was intact, as demonstrated by gel electrophoresis and was suitable for downstream molecular applications, including RT-PCR and qPCR.     

Chemical Analysis of the Lamella Walls of Agaricus bisporus Fruit Bodies

Purified lamella wall fragments of Agaricus bisporus fruit bodies were analyzed and shown to consist of neutral sugars (46.5%), hexosamines (31.7%), proteins (9.5%), some lipid material (10.0%), and ash (1.4%). The cell walls were fractionated on the basis of their polysaccharide solubility in water and alkaline solutions. The isolated fractions, using methylation analysis, exhibited striking chemical structural differences compared with the same fractions obtained from the corresponding vegetative cells and fruit bodies (stipe and pileus) walls. The structural differences detected in the wall seem to correspond to the ultimate differentiation of the mycelium inside the fruit body of A. bisporus. Received: 30 November 1998 / Accepted: 29 January 1999     

Modifications du rythme de fructification de Coprinus congregatus par le dépôt de carpophores mûrs exogènes à la surface des cultures

Changes in Fruiting Rhythm of Coprinus congregatus by Addition of Exogenous Mature Sporophores on the Surface of Cultures. Researches were performed over the regulating effect of mature sporophores on the fruiting rhythm of Coprinus congregatus Bull. ex Fr. Placing exogenous mature fruit-bodies on the vegetative mycelium before the accomplishment of the first flush gives an abundant and perfectly synchronized fructification for this flush. But the fruiting rhythm is then disturbed: compared to control cultures, less sporophores are produced in the following flushes, and, in most cases, the cultures are finally characterized by fewer flushes than the controls. The nature of the substance(s) hypothetically produced by mature sporophores, which may regulate the production of other fruit-bodies, and, correlatively, the fruiting rhythm, is as yet unknown.     

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.     

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.     

Efficiency of Countercurrent Distribution, Sephadex G-10, and Silicic Acid Partition Chromatography in the Purification and Separation of Gibberellin-like Substances from Plant Tissue

The effectiveness of countercurrent distribution, Sephadex G-10column chromatography and silicic acid partition column chromatographyin the purification of gibberellin-like substances from extractsof etiolated Phaseoltu multiflorus seedlings, and elongatingvegetative shoots of Arizona cypress (Cupressus arizonica Greene)and coastal Douglas-fir (Pseudotsuga menziesii var. menziesii)was followed by the use of the barley half-seed -amylase bioassayand dry-weight measurements. Countercurrent distribution resultedin a 10- to 30-fold reduction in the dry weight of the acidic,ethyl acetate-soluble fraction. Sephadex G-10 column chromatographyfurther lowered the dry weight by about two-thirds. Silicicacid partition column chromatography separated gibberellin-likesubstances from each other and again reduced the dry weight.Enhancement of bioassay activity was noted at each step of thepurification procedure. It is concluded that the above proceduresconstitute useful and efficient tools for the initial purificationof gibberellin-like substances from plant tissue.     

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.     

Changes in Endogenous Gibberellin-like Substances in the Vegetative Shoot and Inflorescences in Solanum sisymbrifolium Lam. in Relation to Potassium Content of the Plant

Several acidic gibberellin-like substances were detected inmethanolic extracts from the vegetative shoot and inflorescencesof S. sisymbrifolium by thin layer chromatography and two bioassays.Plants with a relatively low but not a deficient content ofpotassium were shown to contain higher levels of the GA-likesubstances in both tissues than those having a higher K content.Also qualitative changes in the spectrum of the biologicallyactive substances are affected by varying the K content of theplant. The changes in the GA-like substances are found to correlatewith the previously reported morphogenetic effects induced byvarying the K content.