Conditions of cultivation,composition, and biological activity of mycelium of <Emphasis Type="Italic">Flammulina velutipes</Emphasis> (Fr.) P. Karst

A study is made on a strain of higher basydiomycete Flammulia velutipes (Fr.) P. Karst. The conditions of maximum biomass production by Flammulia velutipes were studied. Soluble and insoluble fractions were isolated from mycelium. The composition of cultured mycelium and aqueous extracts from mycelium were investigated. These objects mainly contained carbohydrates (65.3 and 84.0% in insoluble and soluble fractions, respectively, and 56% mycelium), proteins (7.5–10.0% in fractions and 17.5% in mycelium), as well as an insignificant amount of mineral substances. The main carbohydrate component of fractions was glucose (53.6–78.8%); galactose and mannose were also present, as well as fucose and xylose in insignificant amounts. The aqueous extracts from mycelium demonstrated immunomodulating activity. They rendered a stimulating effect on the functional activity of macrophages—central cells of the reticluoendothelial system. The soluble fraction had a more pronounced effect than the insoluble fraction.     

Agrobacterium tumefaciens-Mediated Transformation of the Vegetative Dikaryotic Mycelium of the Cultivated Mushroom Flammulina velutipes

Agrobacterium tumefaciens was used to transform the vegetative dikaryotic mycelium of Flammulina velutipes using a hygromycin B resistance gene as selectable marker. The gene coding for urogen III methyltransferase (cob) was introduced into F. velutipes dikaryotic cells. The resulting transformant cells generated a bright red fluorescence, indicating that cob is promising as a reporter gene in F. velutipes.     

Influence of <Emphasis Type="Italic">Flammulina velutipes</Emphasis> mycelia culture conditions on antimicrobial metabolite production

Enokipodins A, B, C, and D are α-cuparene-type sesquiterpenoids antimicrobial metabolites produced in the stationary stage of Flammulina velutipes mycelia development in malt extract broth. This study assessed the influence of nutritional and environmental factors on F. velutipes mycelia culture for the production of these metabolites. The mycelia growth and antimicrobial activity were assessed by determining dry matter and the diffusion in agar method, respectively. The best F. velutipes mycelia growth was observed in dextrose potato broth, and greater antimicrobial metabolite production occurred in complete Pontecorvo’s culture medium. Environmental modifications, such as a rise in temperature from 25° to 37°C on the 15th day of F. velutipes mycelia culture in malt extract and peptone broth, also optimized antimicrobial metabolite production. The metabolites produced in these treatments were correlated with the enokipodins A and B in thin-layer chromatography (TLC) and the antifungal activity test by TLC bioautography. This study showed that there was no correlation between biomass production and antimicrobial metabolite production, but there may be a correlation between culture medium composition and enokipodins biosynthesis.     

Lipogenesis in the Basidiomycetes Pleurotus ostreatusand Flammulina velutipes Cultivated on Different Media

The compositions of free fatty acids (FA) in the mycelia of oyster cap (Pleurotus ostreatus (Jacq. ex Fr.) Kumm.) and honey mushroom (Flammulina velutipes (Curt. ex Fr.) Sing.) and the effect of mycelium cultivation conditions on the composition and proportions of individual FA were investigated. Palmitic and linoleic acids were found to be major acids produced byP. ostreatus growing on solid agar medium and in a submerged culture with a synthetic medium. The composition of minor FA in P. ostreatuswas dependent on cultivation conditions. Surface cultivation of its mycelium yielded pentadecanoic, octadecenoic, and stearic acids. Submerged cultivation additionally yielded undecanoic, myristic, hexadecenoic, and lignoceric acids. The composition of free FA in F. velutipesshowed no significant differences from that of P. ostreatus. Variation in the C/N ratio in the cultivation medium affected both the FA composition in P. ostreatus and F. velutipes and the relationship between saturated and unsaturated FA.     

Polyol metabolism in the mycelium and fruit-bodies during development ofFlammulina velutipes

Changes of polyol contents in the mycelium and fruit-bodies ofFlammulina velutipes were measured. The results suggested that arabinitol is accumulated in the fruit-bodies as the end-product after its translocation from the mycelium, while mannitol in the fruit-bodies is converted into fructose by the action of mannitol dehydrogenase (MDH). The development of fruit-bodies was promoted by feeding of mannitol to the mycelial colony. A¹⁴C tracer experiment indicated that half of mannitol translocated from mycelium to fruit-bodies was utilized for fruit-body development. NAD-linked MDH andd-arabinitol dehydroganase (D-ADH) were detected in both mycelium and fruit-bodies. The activities of MDH and ADH in the mycelium reached their maximum levels in the inital stage of fruit-body development and decreased thereafter. In contrast, the activity of MDH in the fruit-bodies showed a peak in the middle stage of development. The activity of ADH in the fruit-bodies was less than half of that of MDH. MDH showed a lower Km value for mannitol (1.3 ×10⁻³M) than for fructose (6.0×10⁻² M). The Km value of ADH for arabinitol was extremely high (1.3×10⁻¹M).     

Mitochondrial morphogenesis and ultrastructure of basidiomycetes from genera Agaricus and Pleurotus

Supravital study of rhodamine-stained mitochondria in cells of aerial and submerged micelium of 31 strains from 9 species from genus Agaricus (A. arvensis Schaeff., A. bisporus (Lange) Imbach, A. bitorquis (Quel.) Sacc., A. campestris L., A. excellens, (F.H. Müller) F.H. Müller, A. macrocarpus (F.H. Müller) F.H. Müller, A. silvaticus Schaeff., A. silvicola (Vittad.) Peck, A. xanthodermus Genev) and of 2 strains from 2 species from genus Pleurotus (P. ostreatus (Jacg.) P. Kumm., P. pulmonarius (Fr.) Quel.) was carried out. Mitochondrial morphogenesis in micelial cells of species from Agaricus and Pleurotus genera has many common features in distribution of mitochondria in micelial cells both under the favorable growth conditions and during chondriom reorganization (fission and fragmentation of mitochondria to small subunits) under unfarovable growth conditions and during aging. During the balanced growth of mycelium of heterokaryotic strains from genera Agaricus and Pleurotus for 7–14 days on agar media as well as during cultivation of mycelium of oyster mushroom and several strains of field mushroom in submerged culture, distribution of mitochondria by the type 1 (small granular mitochondria in the apical zone—1, long rod-like mitochondria in the subapical zone—2 and short rod-like and granular mitochondria formed as a result of fragmentation of rod-like mitochondria in mature mycelium cells—3) was observed. Under unfarovable growth conditions (starvation), in mycelium cells of homokaryotic strains of champignon, during long cultivation of all strains and species from the studied genera and during cultivation on liquid wort, for majority of champignon, distribution of mitochondria by the type 2 was observed (sphere-like mitochondria in all cells of the studied mycelium zones). Mitochondrial profiles at ultrastructural level had specific features, such as associations of the outer mitochondrial membrane with cytoplasmic ribosomes and changes in cristae structure. The preliminary test for apoptosis-like phenotype of the submerged mycelium cells of Bs94 champignon that hardly grows in the submerged culture gave positive result. Thus, mitochondrial morphogenesis in mycelium cells of Agaricus and Pleurotus species under different conditions and terms of cultivation occurs similarly and is determined by a complex of physiological and biochemical processes reflecting the state of mycelium cells.     

Polysaccharide composition of mycelium and cell walls of the fungus <Emphasis Type="Italic">Penicillium roqueforti</Emphasis>

Preliminary data on the polysaccharide composition of mycelium and cell walls of the fungus Penicillium roqueforti grown by the method of submerged cultivation have been obtained. Mild acid hydrolysis of both mycelium and cell walls results in formation of glucose, mannose, and galactose, while the treatment with acid under severe conditions results in formation of glucosamine, a product of chitin hydrolysis, the content of which is 19% in the cell walls. Several polysaccharide fractions were isolated from mycelium by successive extraction with hot water and 1 M NaOH at room temperature; their monosaccharide composition was characterized. The main fraction extracted by alkali, according to the data of NMR spectroscopy, mass spectrometry, and the chemical methods of structural analysis, is a linear α-D-glucopyranan, where the blocks of (1 → 3)-bound glucose residues are linked by single bonds (1 → 4). Water-soluble polysaccharides contain the linear blocks of (1 → 5)-bound residues of β-galactofuranose, most probably attached to the mannan core. The findings are of interest for chemotaxonomy of Penicillium fungi.     

Effect of seed-specific expression of the <Emphasis Type="Italic">ipt</Emphasis> Gene on <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> L. seed composition

A transgenic approach to manipulation of endosperm development has been investigated. Nicotiana tabacum cv. Xanthi, an endosperm-containing dicotyledon, has been used as a model plant and the 2.6 kb wheat high molecular weight (HMW) glutenin subunit 12 promoter has been used fused either to the gus reporter gene (HMWgus construct)—to study promoter characteristics—or to the Agrobacterium ipt gene—to study the effect of cytokinin (CK) over-expression on assimilate accumulation in the seed. In transgenic tobacco the promoter:gus fusion showed that HMW is an endosperm-specific promoter with maximum expression 20 days after anthesis (DAA), corresponding to the mid to late stages of seed development. Transgenic plants containing the HMWipt construct showed no morphological abnormalities but they had an average increase in seed weight and total ethanol-insoluble carbohydrates and protein content of 8.1%, 7.0% and 8.3%, respectively. SDS PAGE analysis demonstrated that the effect on protein accumulation was non-specific. The highest values of the parameters analysed correlated with moderate increases in the levels of biologically active CKs. These results suggest that ectopic expression of small amounts of CKs can be used to increase storage assimilate accumulation without a detrimental effect on development.     

Comparative analysis of respiratory activity in the wild type strain of <Emphasis Type="Italic">Neurospora crassa</Emphasis> and its photoreceptor complex mutants

Cell respiratory activity of protoplasts obtained from the wild type of Neurospora crassa and photoreceptor complex WCC—white collar 1 (wc-1) and white collar 2 (wc-2)—mutants of Neurospora crassa strains was investigated. Respiration inhibition by KCN in the presence of 25 mM succinate was similar in all strains and did not exceed 83–85% against control. The significant induction of KCN-resistant respiratory pathway occurred under 1% glucose oxidation in wc-1 and wc-2 mutants if compared with the wild type strains. The inhibitors of the main (cytochrome) pathway of electron transfer in mitochondria—1 mM KCN and antimycin A (4 μg/ml)—blocked the respiration rate of the protoplasts from N. crassa wild type by 75%, while the cell respiration of wc-1 and wc-2 strains was suppressed by approximately 50%. The specific inhibitor of alternative oxidase—10 mM salicylhydroxamic acid (SHAM)—in combination with the blockers of mitochondrial electron transfer chain caused the total suppression of respiratory activity of protoplasts in all studied strains. It is supposed that an increase of KCN-resistance in WCC mutants under glucose oxidation is connected with alternative oxidase activation as the result of failure in reception and signal transduction of active oxygen species.     

Effect of organic and inorganic toxic compounds on luminescence of luminous fungi

The possibility of the development of the solid phase bioluminescent biotest using aerial mycelium of luminous fungi was investigated. Effect of organic and inorganic toxic compounds (TC) at concentrations from 10⁻⁶ to 1 mg/ml on luminescence of aerial mycelia of four species of luminous fungi—Armillaria borealis (Culture Collection of the Institute of Forest, Siberian Branch, Russian Academy of Sciences), A. mellea, A. gallica, and Lampteromyces japonicus (Fungi Collection of the Botanical Institute, Russian Academy of Sciences)—has been studied. Culture of A. mellea was shown to be most sensitive to solutions of the model TC. It was demonstrated that the sensitivity of the luminous fungi is comparable with the sensitivity of the bacteria that are used for environmental monitoring. Use of the aerial mycelium of luminous fungi on the solid support as a test object is a promising approach in biotesting for the development of continuous biosensors for air monitoring.     

Distance-related semi-quantitative estimation of the extramatrical ectomycorrhizal mycelia of <Emphasis Type="Italic">Cortinarius obtusus</Emphasis> and <Emphasis Type="Italic">Tylospora asterophora</Emphasis>

Extramatrical mycelia of ectomycorrhizae (ECM) can markedly differ in their density, organization and extension as well as in their biomass. As these mycelia are very important for nutrient uptake, are sinks for carbohydrates and probably act as potential agents to interfere with mycelia of other ECM, a method allowing calculations of density, extension and amount of extramatrical mycelium could further ecological studies at different scales. As a first step synthesized ECM of Cortinarius obtusus and Tylospora asterophora, which represent two different ‘exploration types’ — T. asterophora belongs to the 'short distance exploration type’ and C. obtusus to the ‘medium distance exploration fringe subtype’ — were compared with respect to their extramatrical mycelia. To investigate the mycelial distribution in detail, flat rhizotrons filled with peat were used to synthesize the ECM on roots of Picea abies (Norway spruce). As both species form white mycelia, they contrast conspicuously to the dark brown peat substrate. This made it favourable to use CD-camera photographs and a consecutive image analysis of the bright mycelium in comparison to the dark background. For that, nine distance areas have been defined as the regions between nine concentric lines placed around the mantle surface of the ectomycorrhizal root (EMR). The distances between these lines were fixed in a regular manner as the square multiples of roughly one diameter of the EMR (0,27 mm). This resulted in lines representing distances of 1, 4, 9, 16, 25, 36, 49, 64, and 81 multiples of the EMR diameter. The distance of 81 EMR diameters equals roughly a real distance of app. 20 mm from the surface of the EMR. The studies showed highly significant differences in mycelial density between the four innermost distance areas, i. e. areas up to a distance of 16 EMR diameters. This applied for both species. Significant differences were found in the same areas when both species were compared. It was shown that C. obtusus still forms a considerable amount of mycelium beyond the 49 distance line, where the substrate was covered by 15–20 %. However, a limited data set did not allow any statistical calculations at these positions. In contrast to C. obtusus, the amount of extramatrical mycelium of T. asterophora decreased more rapidly with increasing distance. Mycelium could not be detected with this method beyond the 49 distance line. We conclude that these two species differ considerably in the amount and extension of their mycelia and this may be in line with their assignment to the respective exploration types. Further studies are needed to substantiate these results with additional species representing the full set of known exploration types. Envisaged correlation studies of these results with mycelial biomass by direct measurement of hyphal length or via quantitative PCR, can possibly result in an estimation of extramatrical mycelia of ECM in natural soil.     

Alterations in the composition of membrane glycero-and sphingolipids in the course of <Emphasis Type="Italic">Flammulina velutipes</Emphasis> surface culture development

Qualitative and quantitative characteristics of the alterations in the lipid composition of the membrane of the basidial fungus Flammulina velutipes in the course of surface culture development were investigated. Modifications of the lipid composition were shown to be timed to specific ontogeny stages, such as changes in the growth rate of the colonies, the appearance of differentiated vegetative cells, and the formation of generative structures. A slowdown of growth correlated with an alteration in the ratio of major classes of phospholipids, namely, with a decrease of phosphatidylcholine relative content and an increase in phosphatidylethanolamines. The differentiation of vegetative cells of the mycelium proceeded along with modifications of molecular composition of glycoceramides. In the course of the first week of growth, the surface culture of F. velutipes produced monohexosylceramides with epoxidized methyl sphingadienine as a sphingoid base. Later on, along with culture growth and specialization of mycelium cells, molecular species with methyl sphingadienine, common for basidiomycetes, start to prevail among the fungal glycoceramides. The formation of fruit bodies is accompanied by enrichment of molecules of phospholipids, mainly, the phosphatidylcholines, with unsaturated fatty acids.     

Translocation of Phosphorus-32 in Sporophores of Collybia velutipes

Phosphorus-32 was absorbed by the mycelium of Collybia velutipes and translocated to the pilei of intact sporophores within 1.5 hours. In the stipes upward translocation occurred first in the peripheral hyphac followed by movement of the isotoipe into the central hyphac. After 3.0 hours hyphae in the two regions were about equally labelled with the isotope. Except for very minor differences hyphal was the same in the peripheral and central portions of the stipe.     

A possible role of the key enzymes of the glyoxylate and gluconeogenesis pathways for fruit-body formation of the wood-rotting basidiomycete Flammulina velutipes

 Biochemical roles of the representative enzymes involved in carbon metabolism of glucose were investigated in relation to the fruit-body formation of the basidiomycete Flammulina velutipes. Changes in specific activities of the enzymes of the tricarboxylic acid (TCA) cycle and glyoxylate (GLOX) and gluconeogenesis pathways were measured at different stages of development of the fungus. The enzyme activities of malate synthase (MS) and fructose bisphosphatase (FBP) as the key enzymes for the GLOX-gluconeogenesis pathways increased in mycelia during the fruit-body formation. The activities of isocitrate dehydrogenase (IDH) for the TCA cycle and NADP-linked glutamate dehydrogenase (GLTDH (NADP)) for glutamate synthesis increased more markedly. Moreover, the mycelial mat of the cultures producing fruit bodies yielded greater enzyme activities of isocitrate lyase (ICL), MS, FBP, and IDH than that of the cultures that did not produce fruit bodies. These results suggest that the GLOX-gluconeogenesis pathways as well as the glutamate synthesis have a strong correlation with the fruit-body formation of F. velutipes. Received: January 22, 2002 / Accepted: May 10, 2002     

Changes in endogenous cytokinin profiles in micropropagated <Emphasis Type="Italic">Harpagophytum procumbens</Emphasis> in relation to shoot-tip necrosis and cytokinin treatments

Changes in cytokinin (CK) profiles and their physiological implications in micropropagated Harpagophytum procumbens [(Burch.) DC. ex Meisn.] tissues in relation to shoot-tip necrosis (STN) and CK treatments were studied. Total CK content was quantified in benzyladenine (BA)-treated necrotic and normal plantlets and in plantlets treated with the CKs BA, meta-topolin (mT) and meta-topolin riboside (mTR) with and without the auxin indole-3-acetic acid (IAA). Generally necrotic shoots yielded more total CK compared to normal shoots. Cytokinin accumulation was higher at the basal section (basal > middle > top). Further analysis of the CKs based on structural and functional forms revealed excessive accumulation of 9-glucosides (deactivation products—toxic metabolites) and limited amounts of O-glucosides (storage forms—re-utilizable) in necrotic and BA-treated shoots compared to normal and topolin-treated cultures. The addition of IAA enhanced the formation of 9-glucosides in BA-treated cultures but reduced it in topolin-treated cultures. The symptom of STN could therefore be attributed to conversion of active cytokinins to other forms such as 9-glucosides which are neither active nor reversibly sequestrated to active forms. Literature shows that metabolites like 9-glucosides of BA have a detrimental effect in plant tissue culture.     

Characterization of proteins expressed abundantly in the fruit-body ofFlammulina velutipes

Chronological changes of protein expression in the vegetative mycelium ofFlammulina velutipes and expression of these proteins in the fruit-body were investigated by two-dimensional polyacrylamide gel electrophoresis. Four proteins (FBA 1-4) expressed abundantly in the fruit-body were found to have different expression patterns in the vegetative mycelium after the fruiting treatment. FBA 1-4 had similar amino acid sequences and displayed a high similarity with the deduced amino acid sequence of theC1 cDNA, which has an Arg-Gly-Asp (RGD) cell-attachment sequence. This suggests that FBA 1-4 may have cell-to-cell attachment activity.     

Biomass production of<Emphasis Type="Italic">Anoectochilus formosanus</Emphasis> hayata in a bioreactor system

We investigated the factors that affect biomass production fromAnoectochilus formosanus in a bioreactor system. Those factors included inoculum size, initial sucrose concentration, media supplements, photosynthetic photon flux density (PPFD), and cuIturing methods. An inoculum size of 8 g L⁻¹ was most suitable for shoot proliferation; biomass accumulation was optimized when the medium was supplemented with 3% sucrose compared with sucrose-free media or those containing concentrations of 6% or 9%. This accumulation also was enhanced under a PPFD of 50 μmol m² s⁻¹. Likewise, the addition of coconut water (50 mL L⁻¹) plus activated charcoal (0.5 mg L⁻¹) to our Hyponex medium proved most beneficial. Comparative studies among three bioreactor systems — continuous immersion, raft (net), and temporary immersion (the ebb and flood system) — revealed that shoot proliferation and biomass accumulation were more efficient when culturing was performed under continuous immersion.     

Polysaccharides from mycelium of the fungus <Emphasis Type="Italic">Cunninghamella japonica</Emphasis>

Preliminary data on the polysaccharide composition of mycelium of the fungus Cunninghamella japonica (synonymous with C. echinulata) grown by the method of submerged cultivation were obtained. Mild acidic hydrolysis of mycelium resulted in the formation of glucose, mannose, and galactose; while the treatment with acid under drastic conditions afforded glucosamine as a product of hydrolysis of chitin and chitosan, their total content was about 35%. Several polysaccharide fractions were isolated from mycelium by successive extraction with hot water, 2% aqueous NaOH, and 10% AcOH; their monosaccharide composition was characterized. The yield of chitosan extracted with AcOH was insignificant. Additional purification of the fraction obtained after extraction with alkali afforded polysaccharide which was a linear (1 → 3)-α-D-glucopyranan according to the data of NMR spectroscopy and the chemical methods of structural analysis. The presence of this polysaccharide, as well as a low content of chitosan and polyuronides, distinguishes the studied strain C. japonica from most of the known Mucorales.     

Lead and cadmium-induced oxidative stress impacting mycelial growth of <Emphasis Type="Italic">Oudemansiella radicata</Emphasis> in liquid medium alleviated by microbial siderophores

In this study, the effects of siderophores produced by six bacteria on mycelium growth, Cd and Pb accumulation, lipid peroxidation, protein content and antioxidant enzyme in Oudemansiella radicata were investigated in Cd and Pb-containing liquid medium. The results showed that inoculation with siderophore-containing filtrates (SCF) partly enhanced the growth of O. radicata after 15 days, with 0.8–32.4% biomass increase for Cd and 0.7–20.8% for Pb compared to control(s), which lacked siderophore. The maximum enhancement for accumulation were found to be confined to Bacillus sp. FFQ2(s) (26.5%) for Cd and Pseudomonas sp. CY63(s) (158.9%) for Pb. A significant decrease in MDA content indicated that lipid peroxidation in O. radicata was alleviated by siderophores. Besides, antioxidant enzyme SOD and POD activities also displayed obviously decrease in SCF-treated mycelium compared to control(s) treatment, while CAT activity did not present significant change. Protein level in O. radicata treated by SCF increased from 0.3 to 138.0% for Cd and from 10.9 to 107.1% for Pb compared to control(s). Therefore, the present work suggests that microbial siderophores can reduce the toxicity of metals to mycelium and then alleviate heavy metals-inducing oxidative stress in O. radicata.