Fruit-body production of test cultures ofFlammulina velutipes preserved for seven years by freezing at three different temperatures

Two strains ofFlammulina velutipes were cultured on PDA plates, and mycelial disks punched out using a cork borer were used for preservation. Five disks of a strain were put into a vial containing one of three cryoprotectants, 10% glycerol, 5% DMSO or 10% polyethylene glycol. Vials were then stored for 7 yr at −20°C, −85°C or liquid nitrogen temperature. The mycelial growth on PDA plates of the cryopreserved mycelial disks, as well as the usual subcultures, were tested two times. After the second test, spawns were prepared for fruit-body production tests by bottle cultivation from selected plates of the second growth tests. The yields of fruit-bodies varied among the cultures derived from the mycelial disks of the same strain preserved under different conditions. Variation in yields was observed even among the mycelial disks preserved at liquid nitrogen temperature, although the range of yield variation was narrower. The yield variation was obvious for the cultures which showed large retardation in the growth test. Four mycelial disks out of the six preserved at −20°C showed higher yields than those preserved at other temperatures. Among the cultures derived from strain FMC224, the control cultures preserved by subculture showed the lowest yield.     

Metabolic function of glycogen phosphorylase and trehalose phosphorylase in fruit-body formation ofFlammulina velutipes

Glycogen phosphorylase in the vegetative mycelium ofFlammulina velutipes converts glycogen to α-glucose 1-phosphate (G1P) in the colony during fruit-body development. Glycogen may contribute to the synthesis of trehalose as the starting material in the vegetative mycelium during the fruiting process of the colony, and the trehalose produced is translocated into the fruit-bodies as the main carbohydrate substrate for their development. Trehalose phosphorylase activity in the vegetative mycelium was at a relatively high level until fruit-body initiation, suggesting the turnover of this disaccharide during the vegetative stage of the colony development. Trehalose phosphorylase activity in the stipes showed a peak level at the early phase of fruit-body development, suggesting the continuing phosphorolysis of trehalose by this enzyme. The stipes also showed a high specific activity of phosphoglucomutase at a sufficient level to facilitate the conversion of G1P to α-glucose 6-phosphate (G6P). In the pilei a large amount of G1P remained until the growth of the fruit-bodies ceased. Trehalase activities in the stipes and pilei were at a very low level, and this enzyme may not contribute to the catabolism of trehalose in the fruit-body development.     

Chitinolytic properties of Streptomyces lividans

Streptomyces lividans TK24, an established host for genetic and molecular studies in actinomycetes, is able to use chitin as sole carbon and nitrogen source. Extracellular chitinase and N-acetyl--d-glucosamidinase (chitobiase) activities were detected in liquid cultures. Chitinase production was inducible by chitin and its low molecular weight derivatives. Low levels of chitinase were also produced in the absence of chitin. Production of extracellular N-acetylglucosaminidase was correlated with the beginning of the stationary phase of growth and was independent of the presence of chitin. Beside highly N-acetylated chitin, supernatants of chitin-induced cultures were able to hydrolyse chitosans with a wide range of degrees of N-acetylation.Abbreviations MS minimal salts - GlcNAc N-acetyl--d-glucosamine - pNP-GlcNAc p-nitrophenyl-2-acetamido-2-deoxy--d-glucopyranoside - d.a. degree of N-acetylation - TLC thin-layer chromatography     

Cultivation of the hyperthermophilic archaeonSulfolobus solfataricus in low-salt media

Two low-salt complex media, bactopeptone and desalted yeast extract, were used for high density cultivation of the hyperthermophilic archaeonSulfolobus solfataricus (DSM 1617). Bactopeptone, which has low mineral ion content among various complex media, was good for cell growth in batch cultures; the maximal cell density in bactopeptone was comparable to that in yeast extract. Howver, cell growth was rather poor when bactopeptone was added by the fed-batch procedure. Since several vitamins are deficient in bactopeptone, the effect of vitamins on cell growth was examined. Among the vitamins tested, pyridoxine was found to improve the growth rate ofS. solfataricus. To reduce the growth inhibition caused by mineral ions, yeast extract was dialyzed against distilled water and then fed-batch cultures were carried out using a feed medium containing desalted yeast extract. Although the concentrations of mineral ions in yeast extract were significantly lowered by the dialysis procedure, fed-batch cultivation with desalted yeast extract was unsatisfactory. To examine whether low molecular weight solutes in yeast extract are crucial for cell growth, we investigated the effect of trehalose, a most abundant compatible solute in yeast extract, on the growth pattern. Cell densities were increased and the length of the lag phase was markedly shortened by the presence of trehalose, indicating that trehalose plays an important role in the growth ofS. solfataricus.     

Cultural properties of a luminous mushroom,Mycena chlorophos

Cultural conditions on mycelial growth and fruit-body formation ofMycena chlorophos were studied. The optimum temperature of the mycelial growth was 27°C and the optimum initial pH of medium was 4.0. Peptone agar medium was suitable for the spawn culture. Compost medium containing rice bran at 10% (fw/fw) was appropriate for fruitbody formation in the Petri dish. Light was essential for initiation of primordia, and low-temperature treatment induced fruit-body formation effectively. The optimum conditions for fruit-body formation were found to be the cultivation at 27°C for 4 wk and continued cultivation for 3 wk under illumination at an intensity above 0.2 lx and at 21°C after casing with moist compost powder. In the fruit-bodies obtained, the maximum photosensitive wavelength of luminescence was 522 nm and the optimum temperature for emission was 27°C. The luminescence of a fruit-body was observed for about 3 d consecutively at 21°C.     

Factors affecting growth and urease production by Trichophyton spp.

Among Trichophyton spp. examined for urease production, T. rubrum was negative, whereas T. mentagrophytes appeared to be the most active species. Urease was not detected in cell-free culture fluids of the tested fungi. The endocellular urease of the test fungi was essentially constitutive. Moreover, addition of urea to the growth medium of these organisms markedly inhibited their mycelial biomass and ureolytic yield. Environmental factors showed variable effects on the test fungi and there was no correlation between mycelial growth and urease activity of these fungi.     

碳源对玫烟色虫草菌丝生长、芽生孢子产生及其耐热性的影响

提高虫生真菌孢子应对热胁迫的能力是生防菌应用研究的关键,为研究菌丝培养阶段碳源对玫烟色虫草Cordyceps fumosorosea IF-1106耐热性的影响,选择了麦芽糖、可溶性淀粉、蔗糖、葡萄糖、果糖、海藻糖为碳源的培养基对玫烟色虫草IF-1106进行液体培养,评估了不同碳源条件下菌丝的生长、产孢及所产芽生孢子的耐热性。结果表明,在菌株培养阶段,培养基中碳源的种类及浓度对菌丝产量、产孢量及所产芽生孢子的耐热性有显著影响,其中蔗糖为碳源时,所产芽生孢子的耐热性强,45 ℃热胁迫条件下LT₅₀为1.65 h;蔗糖浓度为40 g/L时,可产生大量耐热芽生孢子,液体培养3 d后产孢量可达3.43×10⁷个孢子/mL。为探索不同培养条件下所产芽生孢子耐热性不同的原因,提取了孢子内的海藻糖并采用离子色谱法对其进行了定量分析,发现耐热性高的芽生孢子胞内海藻糖含量普遍较低,可见海藻糖是与芽生孢子耐热性密切相关的内源物质。综上所述,选择适宜的培养基是调控孢子耐热性的有效途径,本研究为生产高耐热的玫烟色虫草生防制剂提供了有益的指导。     

Fruit-body production of an ectomycorrhizal fungus in genus <Emphasis Type="Italic">Boletus</Emphasis> in pure culture

Mycelial growth and fruit-body production of an ectomycorrhizal Boletus sp. were examined in pure culture. Mycelia of the strain Bo1 grew well on a medium consisting of sawdust and barley grains. Mature fruit bodies bearing basidiospores were produced after incubation at 22°C for 90 days in the dark, followed by incubation at 26°C for 30–46 days under conditions of high humidity and illumination. The addition of porous stone as a casing on the medium increased fruit-body yield. Deposited spores germinated well on an agar medium and formed mycelial colonies, thus completing the life cycle of Bo1 without a host plant and under axenic conditions. The ability of Bo1 to form ectomycorrhizas was confirmed by axenic resynthesis of mycorrhizas on Quercus serrata. Cultured fruit bodies of Bo1 resembled Gyroporus castaneus and Boletus subcinnamomeus, but its taxonomic position was not elucidated at the species level.     

Estimation of fungal biomass in a solid substrate by three independent methods

Summary Dry matter increase of Agaricus bisporus mycelium in liquid culture, was found to be directly proportional to quantities of fungal derived chitin and extracellular laccase for up to 28 days following inoculation. Fungal ergosterol showed a similar relationship to mycelial growth which was sustained for 56 days of culture. In cultures grown on autoclaved rye grain a correlation was found between the three biochemical indices and linear extension of the mycelium. Each method gave a similar biomass estimate for cultures grown on cereal grains.     

Effect of phosphate deficiency on growth and protein profile in three strains ofPholiota nameko

Changes in mycelial dry weight and soluble protein amounts and acid phosphatase activities on a mycelial dry weight basis in the mycelia and culture supernatants during the Pi-supplied (P⁺) and Pi-depleted (P⁻) cultures of three strains ofPholiota nameko were examined. Mycelial dry weights of the three strains were lower in the P⁻ culture than in the P⁺ culture. However, soluble protein amounts in the culture supernatants and acid phosphatase activities in the mycelia and culture supernatants of the three strains were higher on a mycelial dry weight basis in the P⁻ culture than in the P⁺ culture. Total proteins of strains N2 and N4 were analyzed by two-dimensional-PAGE. Comparison of electrophoretograms of the P⁺ and P⁻ cultures showed that many polypeptides in the two strains were induced and secreted by Pi deficiency, but more than half of them were specific to each strain. Activity staining of acid phosphatase also revealed that two isozymes with the same molecular weights in the three strains were induced and secreted by Pi deficiency. Adaptive mechanisms for Pi deficiency in the three strains were discussed.     

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     

Chitin synthetase activity in a developmental mutant of Phycomyces blakesleeanus

Chitin synthetase activity was analyzed in vitro and in vivo in two morphogenetic stages, namely, dormant spore cells and germlings of the wild type strain and the developmental mutant S356 of Phycomyces blakesleeanus. In vitro experiments showed a much higher specific activity in dormant spores of the mutant strain than in those of the wild-type. This difference was restricted to the dormant spore phase since germlings exhibited comparable levels of activity to those detected in the wild-type strain. Although no correlation was observed between chitin synthesis in vitro and in vivo in mutant spores, germination of these cells was accompanied by an earlier expression of chitin synthetase in vivo. Germination of mutant spores in liquid medium produced morphologically aberrant germlings. Contrary to the extended mycelial growth of the wild-type strain in solid medium, the mutant grew with a typical colonial morphology. Results are discussed in relation to the possible basis of the mutant phenotype.     

铁皮石斛醇溶性浸出物和六类物质的动态变化研究

为了解不同生长期铁皮石斛(Dendrobium officinale)化学成分的变化,对茎、叶醇溶性浸出物、多酚、黄酮、三萜类、有机酸、氨基酸、糖含量进行了测定。结果表明,从2015年9月至2016年9月,茎的醇溶性浸出物总量及六类物质含量呈现先下降后上升的变化趋势,在春末夏初时达到最低,而叶片中各成分的变化未体现一致性规律。六类物质中以小分子的糖含量最高,其次是三萜类和有机酸。茎的醇溶性浸出物总量与多糖含量呈显著负相关;而叶的相关性不显著。这些为评估铁皮石斛质量和确定采收期提供科学依据。     

Mycelial growth of strains of the genera <Emphasis Type="Italic">Suillus</Emphasis> and <Emphasis Type="Italic">Boletinus</Emphasis> in media with a wide range of concentrations of carbon and nitrogen sources

Suillus and Boletinus were studied using Ohta medium. In media with glucose or trehalose, all tested strains grew well. With mannose and cellobiose, strains generally grew well, except for one strain of Suillus. Utilization of dextrin and soluble starch differed with each strain, and that of sucrose and glycerol was low for all strains. Utilization of four amino acids, arginine, glutamic acid, aspartic acid, and alanine, was similar to that of ammonium tartrate for Suillus strains, but mycelial growth with amino acids was clearly lower than with ammonium tartrate for the Boletinus strain. The effect of glucose and ammonium tartrate concentrations for nine strains of the genera Suillus and Boletinus was studied with ranges for glucose of 1–100 and 200g/l, respectively, and for ammonium tartrate of 0.2–5 and 20g/l, respectively. Six strains showed maximal growth at a glucose concentration greater than 25g/l, and one strain showed maximal growth at 70g/l. The results indicate that these fungi are adapted to relatively high concentrations of carbon sources. In general, glucose concentration at mycelial growth maximum decreased as ammonium tartrate concentration increased, and at higher concentrations of glucose, mycelial growth decreased more rapidly in higher concentrations of ammonium tartrate.     

轮纹异痂蝗卵的过冷却能力与其体内水分和生化物质含量的关系

【目的】低温是影响昆虫生长发育和存活的关键因子之一。为明确轮纹异痂蝗Bryodemella tuberculatum dilutum卵的抗寒性及其机理, 研究了其过冷却能力与其体内水分及其他抗寒性相关生化物质含量的关系。【方法】采用热电偶法测定了轮纹异痂蝗卵的过冷却点,同时采用烘干法、残余法、氨基酸自动分析仪和高效液相色谱法分别测定了其卵的含水量、脂肪、氨基酸及小分子糖醇的含量。【结果】土壤含水量对轮纹异痂蝗滞育前卵的含水量、脂肪含量（鲜重）及过冷却点有极显著的影响(P<0.01)。随着土壤含水量从4%增加到13%,卵含水量从45.12%上升到55.25%,卵脂肪含量（鲜重）从10.39%下降到9.39%,而过冷却点从-30.11℃上升到-25.69℃。不同发育阶段卵的过冷却点、含水量、脂肪、氨基酸及小分子糖醇含量存在极显著差异(P<0.01)。从卵产下当天至120 d,卵过冷却点从-26.78℃下降至-30.18℃,含水量从51.93%下降至46.69%,脂肪含量分别从9.99%（鲜重）和17.37%（干重）上升至13.92%(鲜重)和25.29%（干重）。滞育卵的过冷却点显著低于滞育前卵的过冷却点。从卵中共检测到17种氨基酸,其中5种与过冷却点存在显著的相关关系(P<0.05)。卵过冷却点随着甘氨酸和脯氨酸含量的升高而降低,而随着胱氨酸、亮氨酸及天冬氨酸含量的增加而升高。随着卵的发育,海藻糖、甘油、肌醇和山梨醇含量逐渐上升,而葡萄糖和果糖含量逐渐下降。在卵发育过程中,海藻糖和甘油的含量显著高于其他4种物质的含量。卵过冷却点与上述6种小分子糖醇均存在显著的相关关系,其中与海藻糖和葡萄糖的相关性最强。过冷却点随海藻糖、甘油、肌醇和山梨醇含量的升高而降低,而随葡萄糖和果糖含量的升高而上升。【结论】轮纹异痂蝗卵在发育过程中,通过降低含水量,积累脂肪、脯氨酸、甘氨酸及海藻糖、甘油、肌醇和山梨醇等抗寒物质,从而提高其过冷却能力。     

虎掌菌菌丝富硒培养的研究

为生产合适的硒源提供一种思路,以菌丝体生物量、含硒量、还原糖、氨态氮和蛋白质为指标,采用四因素三水平正交设计法优化虎掌菌的富硒发酵条件,探讨不同浓度的硒对虎掌菌固体培养菌丝生长和液体培养产生的生物组分的影响。结果表明,高浓度的硒抑制虎掌菌菌丝体生长;正交试验选择不同的衡量指标,由极差分析得出各因素的影响程度大小,结合证实试验得到以还原糖为指标的最优组合为葡萄糖6%(质量分数)、酵母浸膏3%(质量分数)、KH_2PO_4 0.1%(质量分数)、Na_2SeO_3 0.6 mmol/L,其菌丝体生物量和氨态氮含量较高。与对照相比,加硒后虎掌菌发酵液中氨态氮、还原糖和总糖含量显著增加(P0.05);当硒浓度为0.5 mmol/L时,氨态氮、还原糖和总糖含量均达到最高值。菌丝体生物量和可溶性蛋白质分别在硒浓度为0.2 mmol/L和0.4 mmol/L时达到最大值。虎掌菌富硒培养后,发酵液的营养成分含量会发生变化。     

Effects of paclitaxel-producing fungal endophytes on growth and paclitaxel formation of <Emphasis Type="Italic">Taxus cuspidata</Emphasis> cells

Effects of a fungal endophyte, Fusarium mairei, on growth and paclitaxel formation of Taxus cuspidata cells were investigated by adding fungal endophyte culture supernatant (FECS) to suspension cultures of T. cuspidata cells. The main effective chemical responsible for paclitaxel formation in FECS was an exopolysaccharide (EPS) of molecular weight ~2 kDa. FECS fractions except EPS stimulated growth of Taxus cells but had no effects on paclitaxel accumulation. Additionally, elicitation efficiency of FECS based on different culture conditions was studied. EPS content in FECS was related to FECS culture conditions. FECS with long cultivation and high-aeration cultivation contained higher EPS content and resulted in higher paclitaxel yield than that with short cultivation and low-aeration cultivation. The maximum yield of paclitaxel from Taxus cultures, elicited by FECS with 9-day cultivation, was 4.7-fold that of the control cultures.     

Activation of intracellular and extracellular phenol oxidases in photoinduced fruit-body formation ofFavolus arcularius

The activation of intracellular phenol oxidase (PO) was associated with the photoinduced fruit-body initiation inFavolus arcularius. The second activation of PO activity was also photoinduced in the rapidly developing stipe after the formation of pileus primordium under light exposure. However, the activity levels in the pileus and the mycelium remained low even in the cultures exposed to light. The extracellular PO activity in the culture filtrate also appears to be developmentally regulated: it rose sharply after primordium formation in the light, then decreased rapidly during further development of the fruit-body.     

Controlled mycelial growth for kojic acid production using Ca-alginate-immobilized fungal cells

Summary Conidia of Aspergillus oryzae were immobilized in Ca-alginate beads and then incubated in a nutrient medium to yield an immobilized biocatalyst producing kojic acid. The immobilized cell cultures produced kojic acid linearly during cultivation. Regardless of the size of the immobilized particles, there existed an optimal nitrogen concentration for the maximum production rate of kojic acid, at which smaller bead sizes resulted in a higher production rate. When the growth of mycelia were confined within the bead surface and segregated from each other by gel material, they produced kojic acid with maximal catalytic activity and exhibited the highest conversion yield of glucose. The extent of mycelial segregation was especially higher in cultures of smaller bead particles, and the depth of mycelial growth was 150 to 250 m from the gel bead surface in all cultures of different nitrogen concentrations and bead sizes. Therefore, for the maximum expression of catalytic activities of immobilized mycelial cultures, it was found very critical to optimally control the mycelial distribution in gel beads by the culture conditions affecting mycelial growth.     

Growth and macromolecular content of the dimorphic fungus Aureobasidium pullulans and the effect of hydroxyurea and other inhibitors

The growth kinetics and the macromolecular content of the yeast and ethanol-induced hyphal forms of Aureobasidium pullulans were studied. During the morphological transition from yeasts to hyphae, both the protein and RNA content decreased significantly, the mycelial form containing only 76% of the amount of protein in the yeasts, and 38% of the RNA. The DNA was the only component tested whose level increased during the transition. Among several compounds inhibiting macromolecular synthesis, only hydroxyurea showed a remarkable effect on the morphology of A. pullulans, inducing the mycelial morphology. The macromolecular composition of hydroxyurea-treated cultures changed with time in a way similar to that of the ethanol-Tween 80-ammonia medium, and to that of carbon-starved cultures, without ethanol or glucose.