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.     

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.     

Some biological characteristics of the casing soil and their effect during Agaricus bisporus fructification

The intent of this work was to study the biological and biochemical aspects of the casing soil role on every stage of the A. bisporus fructification. We reported the development of mycelial thick threads of A. bisporus in the casing soil. Moreover, we pointed out the protective role of the calcium salts crystals surrounding the hyphae during the incubation stage. On the electron-micrographies, the mycelial aggregates were surrounded by bacteria, and this bacterial growth seemed to be related to the calcium salts crystals disappearance. The positive influence of A. bisporus upon bacterial growth, and vice versal, was confirmed with PETRI dish cultures.     

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.     

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.     

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.     

Effects of the mushroom-volatile 1-octen-3-ol on dry bubble disease

Dry bubble disease caused by Lecanicillium fungicola is a persistent problem in the cultivation of the white button mushroom (Agaricus bisporus). Because control is hampered by chemicals becoming less effective, new ways to control dry bubble disease are urgently required. 1-Octen-3-ol is a volatile that is produced by A. bisporus and many other fungi. In A. bisporus, it has been implicated in self-inhibition of fruiting body formation while it was shown to inhibit spore germination in ascomycetes. Here, we show that 1-octen-3-ol inhibits germination of L. fungicola and that enhanced levels of 1-octen-3-ol can effectively control the malady. In addition, application of 1-octen-3-ol stimulates growth of bacterial populations in the casing and of Pseudomonas spp. specifically. Pseudomonas spp. and other bacteria have been demonstrated to play part in both the onset of mushroom formation in A. bisporus, as well as the inhibition of L. fungicola spore germination. A potential role of 1-octen-3-ol in the ecology of L. fungicola is discussed.     

Evaluation of indigenous potent mushroom growth promoting bacteria (MGPB) on Agaricus bisporus production

Mushrooms such as Agaricus bisporus, are cultivated for food worldwide. Fruit body initiation in Agaricus bisporus is a phase change from the vegetative to the reproductive stage which depends on the presence of a casing layer with particular physical, chemical and microbiological properties. The phase change is achieved practically by environmental manipulation and the presence of naturally occurring bacteria such as Pseuodomonas putida. In this study, 274 individual bacterial isolates were collected by screening the casing layer of 14 edible mushroom farms. The isolates were analysed with respect to biochemical properties, organic and inorganic phosphate solubilization, production of siderophore and growth in the presence of volatile compound of 1-octen-3-ol. It was found that approximately 97% of the strains were able to grow in the presence of 1-octen-3-ol and 36% were able to solubilize phosphorus. Among the isolates, 23 strains were selected as potent mushroom growth promoting bacteria (MGPB) for inoculation of the casing layer. Field experiments using these strains showed various promoting effects on production of mushroom. Finally, 2 strains (strains Bt4 and Ps7) showing the highest increase in A. bisporus production, were characterized as Pseuodomonas putida by molecular methods and identified as the best suited growth promoting inoculants for application in production farms for increasing the mushroom yield.     

The effect of some microphagous saprobic nematodes on mushroom culture

When added to spawned mushroom compost the microphagous saprobic nematodes Pelodera cylindrica, Mesorhabditis spiculigera and Acrobeloides bütschlii did not affect mycelial growth or compost pH. The eelworms multiplied at first but their numbers declined as the mycelium colonized the compost. In contrast, the fungal-feeding Aphelenchoides composticola increased rapidly and destroyed the mycelium. Excess water, and the presence of the fungal competitor of mushroom, Chaetomium olivaceum, allowed some increase of the microphagous forms. Different numbers of Mesorhabditis spiculigera added to the ‘casing’ had no effect on mushroom yield but the mushroom ‘flushes’ seemed less pronounced. Reasons for the failure of the saprobic eelworms to affect mushroom, and the possibility of synergistic pathogenicity by these eelworms and bacteria, are discussed.     

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.     

双孢蘑菇疣孢霉病的发病过程及病原菌的核相研究

【目的】确定有害疣孢霉的传播途径,明确双孢蘑菇受有害疣孢霉侵染后发病症状和微观形态变化,以及有害疣孢霉的核相。【方法】将有害疣孢霉喷施于培养料及覆土材料的不同深度,观察记录双孢蘑菇的发病情况;将有害疣孢霉接种于不同生长阶段的双孢蘑菇子实体,观察记录其发病情况;使用光学显微镜及扫描电镜观察双孢蘑菇子实体受有害疣孢霉侵染前后的形态变化;通过DAPI(4′,6-二脒基-2-苯基吲哚)染色的方法对有害疣孢霉核相进行观察。【结果】将有害疣孢霉接种于培养料及覆土层的不同深度得到双孢蘑菇发病率如下:覆土层表面覆土层中间覆土与培养料交界处培养料中间层;有害疣孢霉可以侵染双孢蘑菇的任意阶段,将其接种于原基直径小于3 mm子实体表面时,得到不能正常分化的"马勃状"组织;对有害疣孢霉的侵染过程进行观察得到:其孢子可粘附于双孢蘑菇表面,并萌发长出芽管,接种处双孢蘑菇表面产生褐色病斑,双孢蘑菇菌丝体发生质壁分离,最后菌丝体膨大,细胞壁变薄甚至溢裂,菌丝体内部中空;有害疣孢霉产生两种类型的分生孢子,Ⅰ类无隔膜含1个细胞核;Ⅱ类具1隔膜含2个细胞核,2个细胞核被隔膜分开;细胞核的第1次有丝分裂发生于分生孢子母细胞中;厚垣孢子由上下2个细胞构成,上胞中含有2个细胞核。下胞含1–2个细胞核。有害疣孢霉的厚垣孢子萌发可产生1–2个芽管,芽管中细胞核的数目不断变化,一般0–2个细胞核。【结论】双孢蘑菇受其侵染后发生显著的细胞学变化;我们对有害疣孢霉做遗传分析时,进行单孢分离需挑取无隔膜的分生孢子为实验材料进行遗传分析。     

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.     

Stimulation of indirubin production by KNO3 depletion in indole-supplemented suspension culture of Polygonum tinctorium

Summary Indole when added at 20 mM to shake flask cultures of Polygonum tinctorium cells on 16th day increased indirubin production from 41 mg/l to 126 mg/l. Use of KNO₃ depleted media stimulated indirubin formation up to 88% and 26% in shake flasks and air-lift bioreactors, respectively.     

Bacteria produce the volatile hydrocarbon isoprene

Various bacterial species, both Gram-negative and Gram-positive, were found to produce the volatile hydrocarbon isoprene (2-methyl-1,3-butadiene). Out of the tested cultures, Bacillus produced the most isoprene. The production of isoprene from bacteria was confirmed by gas chromatography-mass spectrometry. Media and growth effects on isoprene production were investigated: growth in rich media led to higher levels of isoprene than growth in minimal media, and highest isoprene emission rates were seen in log-phase cultures. Temperature profiles for bacterial isoprene production showed an optimum of 45°C and were suggestive of an enzymatic mechanism for isoprene formation.     

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.     

Suppressiveness of Vermicompost against Fusarium Wilt of Tomato

Vermicompost added to various container media significantly inhibited the infection of tomato plants by Fusarium oxysporum f. sp. lycopersici. The protective effect increased in proportion to the rate of application of vermicompost. Every type of container media amended with this substrate, used in the experiments, were suppressive to the pathogen. Vermicompost lost its activity after heating. Sterilized extracts of vermicompost added to potato dextrose agar stimulated the growth of F. oxysporum mycelium. The results indicate that chemical factors in this substrate had no direct inhibiting effect on the fungus. The total number of micro-organisms and populations of antagonistic bacteria and fungi were significantly higher in vermicompost than in the control peat substrate. A biotic nature is suggested for the suppressiveness of the vermicompost.     

New Strategies for Cultivation and Detection of Previously Uncultured Microbes

An integrative approach was used to obtain pure cultures of previously uncultivated members of the divisions Acidobacteria and Verrucomicrobia from agricultural soil and from the guts of wood-feeding termites. Some elements of the cultivation procedure included the following: the use of agar media with little or no added nutrients; relatively long periods of incubation (more than 30 days); protection of cells from exogenous peroxides; and inclusion of humic acids or a humic acid analogue (anthraquinone disulfonate) and quorum-signaling compounds (acyl homoserine lactones) in growth media. The bacteria were incubated in the presence of air and in hypoxic (1 to 2% O₂ [vol/vol]) and anoxic atmospheres. Some bacteria were incubated with elevated concentrations of CO₂ (5% [vol/vol]). Significantly more Acidobacteria were found on isolation plates that had been incubated with 5% CO₂. A simple, high-throughput, PCR-based surveillance method (plate wash PCR) was developed. This method greatly facilitated detection and ultimate isolation of target bacteria from as many as 1,000 colonies of nontarget microbes growing on the same agar plates. Results illustrate the power of integrating culture methods with molecular techniques to isolate bacteria from phylogenetic groups underrepresented in culture.     

Production of Oil-Releasing Compounds by Microorganisms from the Daqing Oil Field,China

Twenty pure cultures isolated from formation waters of the Daqing oil field were studied with respect to their capacity to produce surface-active compounds in media with individual hydrocarbons, lower alcohols, and fatty acids. Aerobic saprotrophic bacteria belonging to the genera Bacillus, Brevibacillus, Rhodococcus, Dietzia, Kocuria, Gordonia, Cellulomonas, Clavibacter, Pseudomonas, and Acinetobacter decreased the surface tension of cultivation media from 55–63 to 28–44 mN/m. Strains of Bacillus cereus, Rhodococcus ruber, andBacillus licheniformis produced biosurfactants most actively. Bacteria of the genera Rhodococcus, Dietzia, Kocuria, and Gordonia produced exopolysaccharides in media with hydrocarbons. Culture liquids of the strains of R. ruberand B. licheniformis exhibited an oil-releasing effect. Thus, the Daqing oil field is inhabited by aerobic bacteria capable of producing effective oil-releasing agents.     

The cel4 Gene of Agaricus bisporus Encodes a β-Mannanase

Mannases have industrial uses in food and pulp industries, and their regulation may influence development of the mushrooms of commercially important basidiomycetes. We expressed an Agaricus bisporus cel4 cDNA, which encodes a mannanase, in Saccharomyces cerevisiae and Pichia pastoris. CEL4 had no detectable activity on cellulose or xylan. This gene is the first isolated from this economically important fungus to encode a mannanase. P. pastoris secreted about three times more CEL4 than S. cerevisiae. The removal of the cellulose-binding domain of CEL4 lowered the secreted specific activity by P. pastoris by approximately 97%. The genomic sequence of cel4 was isolated by screening a cosmid library of A. bisporus C54-carb8. The open reading frame was interrupted by 12 introns. The level of extracellular CEL4 increases dramatically at the postharvest stage in compost extracts of A. bisporus fruiting cultures. In laboratory liquid cultures of A. bisporus, the activity of CEL4 detected in the culture filtrate reached a maximum after 21 days. The levels of CEL4 broadly mirrored the levels of enzyme activity. In the Solka floc-bound mycelium, CEL4 protein showed a maximum after 2 to 3 weeks of culture and then declined. Changes in CEL4 activity during fruiting-body development suggest that hemicellulose utilization plays an important role in sporophore formation. The availability of the cloned gene will further studies of compost decomposition and the extracellular enzymes that fungi deploy in this process.