Physiological and genetic characterization of fluorescent Pseudomonas associated with Cantharellus cibarius

Fluorescent Pseudomonas spp. isolated from fruiting bodies (FB) of Cantharellus cibarius were characterized physiologically and genetically and were compared with fluorescent Pseudomonas from forest soil and with sequences from the GenBank database. Pseudomonas spp. from FB differed physiologically from isolates from soil lacking FB and had some similarities with the strains obtained from soil underneath the FB. Analyses of the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) patterns and partial sequencing analysis of the 16S-rDNA region indicated that the bacteria isolated from these environments were different. However, there was no specific Pseudomonas genotype restricted to the FB environment. Utilization of the reported fungal exudates trehalose and mannitol may explain how millions of bacteria survive in the C. cibarius FB without deteriorating the fungal mycelium. The importance of the metabolic characterization of bacteria and the possible mechanisms involved in the association with C cibarius are discussed. Our study showed that standard processes for bacterial identification, e.g., Biolog and 16S-rDNA are insufficient until databases for different ecosystems are created.     

鸡油菌的25S rDNA部分序列及其系统学意义

菌物分类学家通常根据形态学、解剖学和显微观察特征的分析把鸡油菌类真菌（cantharelloidfungi）置于非褶菌目Aphyllophorales中或在非褶菌目和其他真正的伞菌之间,但由于缺乏足够的科学证据,有关其进化历史及其在担子菌中的系统发育位置等方面的关系等的各种假设,一直都存在着激烈的争论。本文从分子系统学角度出发,对鸡油菌Cantharelluscibarius的255rDNA部分序列进行了测定,并与另外10目的13个担子菌代表种和一个接合菌代表种的255rDNA序列进行比较分析,以接合菌总状毛霉Mucorracemosus为外类群构建出这些担子菌的系统树图,探讨鸡油菌与其他担子菌的关系及其系统学上的意义。从所构建的100多个进化树图来看,鸡油菌几乎总是在树图中最远的分枝的末端,与同担子菌中的各类肉质的伞菌目Agaricales和牛肝菌目Boletales的类群（乳菇属Lactarius、褶孔菌属Phylloporus、鹅膏属Amanita和口蘑属Tricholoma）最接近,其次是同担子菌中的非褶菌目的类群（侧耳属Pleurotus和灵芝属Ganoderma）,然后才是木耳Auricularia等有隔担子菌的类群。用于比较的鸡油菌DNA片段长度最长,有258个碱基,而其他类群则只有214至此241个碱基。本文认为鸡油菌与其他各肉质的伞菌类群的关系相对较密切,但它应该有其相?     

Use of different nitrogen sources by the edible ectomycorrhizal mushroom Cantharellus cibarius

The growth of three strains of Cantharellus cibarius on liquid media containing ammonium, nitrate and bovine serum albumin (BSA) in different combinations was determined. The most readily utilisable source of N was ammonium. BSA utilisation was limited compared with media containing ammonium. Growth on nitrate was also poor, suggesting a limited capacity of C. cibarius to metabolise this nitrogen source. There was some indication of considerable intraspecific variation within C. cibarius in the utilisation of nitrogen sources. Possible links between atmospheric nitrogen deposition and the observed decrease of sporocarp formation by C. cibarius in Europe are discussed. We highlight the potential ecological significance of bacteria associated with C. cibarius which may circumvent the need for fungal extracellular enzymes to access complex nitrogen sources.     

C Nuclear Magnetic Resonance Study of Mannitol Cycle and Trehalose Synthesis during Glucose Utilization by the Ectomycorrhizal Ascomycete Cenococcum graniforme

¹³C nuclear magnetic resonance spectroscopy has been used to follow the utilization of glucose for the synthesis of carbohydrates in the ectomycorrhizal ascomycete Cenococcum graniforme. The fate of ¹³C label was analyzed in vivo and in mycelial extracts. The major carbohydrates produced from [1-¹³C]glucose and [6-¹³C]glucose were mannitol and trehalose. Mannitol was mainly synthesized via a direct route from glucose. Scrambling of the ¹³C label was observed to occur in trehalose during glycolysis. From the analysis of the scrambling patterns, it is concluded that the mannitol cycle was operative and that a large part of the carbon of glucose was used to form trehalose after cycling through the mannitol pool. The activities of NAD-mannitol-l-P dehydrogenase (EC 1.1.1.17) and NADP-mannitol dehydrogenase (EC 1.1.1.138), which participate in the mannitol cycle relative to the activity of glycolytic enzymes, provide evidence that the cycle is important for NADPH production.     

Root influence on in vitro growth of hyphae of the mycorrhizal mushroom Cantharellus cibarius replaced by carbon dioxide

The presence of a living root of tomato ( Lycopersicon esculentum Mill. cv. Panase F) had a strong positive effect on growth in vitro of hyphal fragments of Cantharellus cibarius Fr. This was observed for 12 out of 14 fungal strains tested. Initial re-growth of hyphae was independent of the presence of a root, but for the further development of strong mycelial growth 7 strains completely depended on the presence of a root. Five strains showed a considerably prolonged lag phase in the absence of a root and only 2 strains were independent. The presence of a root had no influence on the specific growth rates. Hyphal fragments of the totally root-dependent strain S1 grew strongly in the absence of a root if they were brought into intimate mutual contact. Factor(s) stimulating growth were apparently produced by both roots and fungus. Volatiles from donor cultures of tomato roots of C. cibarius, Boletus edulis , and Tylopilus felleus stimulated growth of hyphal fragments in root-less receiver cultures. This effect was prevented by the presence of a KOH solution. Donor cultures could be replaced by 0.5% CO₂.     

Carbohydrate and Amino Acid Metabolism in the Ectomycorrhizal Ascomycete Sphaerosporella brunnea during Glucose Utilization : A C NMR Study

Nuclear magnetic resonance spectroscopy was utilized to study the metabolism of [1-¹³C]glucose in mycelia of the ectomycorrhizal ascomycete Sphaerosporella brunnea. The main purpose was to assess the biochemical pathways for the assimilation of glucose and to identify the compounds accumulated during glucose assimilation. The majority of the ¹³C label was incorporated into mannitol, while glycogen, trehalose and free amino acids were labeled to a much lesser extent. The high enrichment of the C1/C6 position of mannitol indicated that the polyol was formed via a direct route from absorbed glucose. Randomization of the ¹³C label was observed to occur in glucose and trehalose leading to the accumulation of [1,6-¹³C]trehalose and [1,6-¹³C]glucose. This suggests that the majority of the glucose carbon used to form trehalose was cycled through the metabolically active mannitol pool. The proportion of label entering the free amino acids represented 38% of the soluble ¹³C after 6 hours of continuous glucose labeling. Therefore, amino acid biosynthesis is an important sink of assimilated carbon. Carbon-13 was incorporated into [3-¹³C]alanine and [2-¹³C]-, [3-¹³C]-, and [4-¹³C]glutamate and glutamine. From the analysis of the intramolecular ¹³C enrichment of these amino acids, it is concluded that [3-¹³C]pyruvate, arising from [1-¹³C]glucose catabolism, was used by alanine aminotransferase, pyruvate dehydrogenase, and pyruvate carboxylase (or phosphoenolpyruvate carboxykinase). Intramolecular ¹³C labeling patterns of glutamate and glutamine were similar and are consistent with the operation of the Krebs cycle. There is strong evidence for (a) randomization of the label on C2 and C3 positions of oxaloacetate via malate dehydrogenase and fumarase, and (b) the dual biosynthetic and respiratory role of the citrate synthase, aconitase, and isocitrate dehydrogenase reactions. The high flux of carbon through the carboxylation (presumably pyruvate carboxylase) step indicates that CO₂ fixation is an important component of the carbon metabolism in S. brunnea, and it is likely that this anaplerotic role is particularly prevalent during NH₄⁺ assimilation. The most relevant information resulting from this investigation is (a) the occurrence of the mannitol cycle, (b) a large part of the trehalose pool is synthesized after the cycling of glucose-carbon through the mannitol cycle, and (c) pyruvate (or phosphoenolpyruvate) carboxylation plays an important role in the primary metabolism of glucose-fed mycelia.     

鸡油菌属的研究概况与展望

鸡油菌属Cantharellus Adans. ex Fr. 在担子菌中是一个具有独立演化路线的类群, 隶属于担子菌门,同担子菌纲, 鸡油菌目, 鸡油菌科。该属全球分布广泛, 目前有65种, 中国曾记载9种。本文回顾了该属的研究历史, 结合自己的研究成果,着重对本属各分类单元的划分和命名, 相近种的区分, 鸡油菌C. cibarius Fr. 和管形鸡油菌C. tubaeformis Fr.: Fr. 复合群以及生态保护和仿生栽培等方面研究中存在的疑问和纷争进行了论述。最后,作者对我国鸡油菌属的生物多样性     

13C nuclear magnetic resonance and gas chromatography-mass spectrometry studies of carbon metabolism in the actinomycin D producer Streptomyces parvulus by use of 13C-labeled precursors.

Fructose and glutamate metabolism was monitored in cell suspensions of streptomyces parvulus by 13C nuclear magnetic resonance. The experiments were performed for cells grown with various 13C sources in a growth medium containing D-[U-13C]fructose, L-[13C]glutamate, or L-[U-13C]aspartate and with nonlabeled precursors to compare intracellular pools in S. parvulus cells at different periods of the cell life cycle. The transport of fructose into the cells was biphasic in nature; during rapid transport, mannitol, fructose, and glucose 6-phosphate were accumulated intracellularly, whereas during the passive diffusion of fructose, the intracellular carbohydrate pool comprised mainly trehalose (1,1'-alpha-alpha-D-glucose). The regulation of fructokinase activity by the intracellular intermediates may play an important role in fructose catabolism in S. parvulus. Transaldolase activity in S. parvulus was determined from the 13C nuclear magnetic resonance labeling pattern of trehalose carbons obtained from cells grown in medium containing either L-[U-13C]aspartate or L-[U-13C]glutamate. Only carbons 4, 5, and 6 of the disaccharide were labeled. Isotopomer analysis of the trehalose carbons led us to conclude that the flux through the reverse glycolytic pathway, condensation of glyceraldehyde 3-phosphate with dihydroxyacetone phosphate, makes at best a minor contribution to the 13C-labeled glucose units observed in trehalose. The pentose pathway and transaldolase activity can explain the labeling pattern of 4,5,6-13C3 of trehalose. Moreover, the transfer of the 13C label of L-[U-13C]aspartate into the different isotopomers of trehalose C4, C5, and C6 by the transaldolase activity allowed us to calculate the relative fluxes from oxaloacetate via gluconeogenesis and through the tricarboxylic acid cycle. The ratio of the two fluxes is approximately 1. However, the main carbon source for trehalose synthesis in S. parvulus is fructose and not glutamate or aspartate. The 13C enrichment and isotopomer population, measured by nuclear magnetic resonance and gas chromatography-mass spectrometry, of the actinomycin D peptide ring enabled us to specify the origins of the five amino acids of actinomycin D. Threonine and proline exhibited isotopomer populations similar to that of the extracellular L-[13C]glutamate, indicating that protein catabolism is the origin of their 13C label, whereas the isotopomer populations of sarcosine and N-methylvaline were similar to those of the new intracellular pool of S. parvulus that originated from D-[U-13C]fructose during the production of actinomycin D.     

Discovery and structural characterization of fucosylated oligomannosidic N-glycans in mushrooms

L-fucose is a common constituent of Asn-linked glycans in vertebrates, invertebrates, and plants, but in fungal glycoproteins, fucose has not been found so far. However, by mass spectrometry we detected N-glycans and O-glycans containing one to six deoxyhexose residues in fruit bodies of several basidiomycetes. The N-glycans of chanterelles (Cantharellus cibarius) contained a deoxyhexose chromatographically identical to fucose and sensitive to α-L-fucosidase. Analysis of individual glycan species by tandem MS, glycosidase digestion, and finally (1)H NMR revealed the presence of L-fucose in α1,6-linkage to an α1,6-mannose of oligomannosidic N-glycans. The substitution by α1,6-mannose of α1,2-mannosyl residues of the canonical precursor structure was yet another hitherto unknown modification. No indication for the occurrence of yet other modifications, e.g. bisecting N-acetylglucosamine, was seen. Besides fucosylated N-glycans, short O-linked mannan chains substituted with fucose were present on chanterelle proteins. Although undiscovered so far, L-fucose appears to represent a prominent feature of protein-linked glycans in the fungal kingdom.     

A ubiquitin-like peptide with ribonuclease activity against various polyhomoribonucleotides from the yellow mushroom Cantharellus cibarius

A peptide, with a molecular weight of 9K and an N-terminal sequence identical to that of ubiquitin, was isolated from fresh fruiting bodies of the yellow mushroom Cantharellus cibarius. The peptide manifested ribonucleolytic activity toward poly A, poly C, poly G, and poly U, with the activity toward the first two polyhomoribonucleotides being higher. Maximal activity toward yeast tRNA was observed at a temperature of 70 degrees C and a pH of 7. The peptide was adsorbed on DEAE-cellulose, CM-Sepharose, and Q-Sepharose. The yield of the peptide was 7mg from 1.8kg mushroom.     

The soluble carbohydrates of Euglena gracilis—their identity and response to increased external solute concentration

Abstract Mannitol and trehalose were the predominant soluble carbohydrates in Euglena gracilis strain z growing heterotrophically in complete darkness or in light. The ratio of trehalose to mannitol was correlated with the water activity of the medium. That is, extracts of Euglena gracilis adapted to grow in media supplemented with either sodium chloride or glucose, thereby reducing the water activity, yielded molar ratios of trehalose to mannitol 10 times greater than extracts of cultures grown under conditions in common usage.     

Multiple component system of sugars and polyols in the overwintering spruce bark beetle, Ips typographus

Overwintering adults of the spruce bark beetle, Ips typographus (L.) showed an unusually complex sugar/polyol cryoprotectant system. The major components of the multiple system were: glucose (177.6 mmolL(-1), March); trehalose (175.0 mmolL(-1), December); sorbitol (147.9 mmolL(-1), January); mannitol (81.2 mmolL(-1), March); and erythritol (40.7mmolL(-1), March) (in the parentheses, the maximum concentrations are shown and the month when they were reached). Other minor components were glycerol, fructose, threitol, myo-inositol, arabinitol and ribitol. Distinct seasonal patterns of accumulation/depletion in various components were found. Glycerol, trehalose and glucose started to accumulate first, during early autumn, when the air temperatures fluctuated between 20 and 0 degrees C, and diapause beetles continued in feeding. Glycerol was depleted, glucose remained stable and trehalose continued in accumulation during late autumn when the temperatures oscillated around 0 degrees C. During early winter severe frosts reaching -20 degrees C came, the beetles terminated their diapause and trehalose was partially depleted, while mannitol, sorbitol, fructose, threitol and erythritol started to accumulate. Cold weather continued also during late winter when the beetles remained quiescent. During this period, trehalose was re-accumulated, threitol and erythritol continued to increase, mannitol remained stable and sorbitol, fructose decreased. All cryoprotectans were finally cleared in the beetles which were spontaneously leaving bark during early spring. The seasonal maximum of total concentration of all cryoprotectants (578.2 mOsmol L(-1)) was reached in March. Such a concentration results in colligative depression of melting point of body fluids down by 1.08 degrees C only. It suggests that the potential cryoprotective effect of accumulated sugars and polyols was related rather to their non-colligative functions.     

Notes nomenclaturales et taxinomiques sur deux espèces françaises de Cantharellus (basidiomycotina)

Cantharellus pseudominimus nom. nov. is proposed as a replacement for C. minimus L. L. Daniel, 1912 (non C. minimus Vaill. ex Roussel, 1795–1796). C. cibarius var. tenuis Romagn. is described and illustrated after revision of the type specimen. As this taxon can not be considered a mere variety of C. cibarius, C. romagnesianus nom. nov. is proposed.     

Cryopreservation of Digitalis lanata Ehrh. cell cultures: Preculture and freeze tolerance

Cryopreservation experiments were performed with Digitalis lanata cell cultures. The main stress was laid on the behaviour of the cells during the preculture period and the capacity of various preculture additives to induce freeze tolerance. The following compounds were used as preculture additives: trehalose, mannitol, sucrose, melibiose, proline, and sorbitol. They are listed in the order of their respective efficiency. Using trehalose, high post-thaw viability rates were achieved and the cells resumed growth after a short lag period. Melibiose was used as a preculture additive for the first time. Its suitability was in the range of that of sucrose. Proline and sorbitol were not able to induce freeze tolerance in Digitalis cells. Cell viability showed a considerable decrease at the beginning of the preculture period. This reduction was found to be transient in the presence of trehalose, mannitol, sucrose, and melibiose. The damaging effects of proline and sorbitol were too severe to be compensated for by the cells. The PAL activity increased markedly in the presence of proline, whereas the trehalose-treated and the control cells behaved nearly identical to one another.     

Compatible solutes of sclerotia of Mycoleptodiscus terrestris under different culture and drying conditions

Mycoleptodiscus terrestris is a plant pathogen which has been shown to be effective in controlling invasive aquatic weeds in inundative biocontrol applications. The preferred propagule for production and application is the sclerotium. In the current study, we evaluated the accumulation of carbohydrates and polyols in the sclerotia of M. terrestris under different culture and drying regimes. The carbohydrates and polyols screened for represent a class of compatible solutes found in fungi. The results show mannitol and trehalose are the major analytes identified in the sclerotia of M. terrestris. We identified higher levels of mannitol and trehalose in liquid-culture produced samples relative to solid-state produced samples. The impact of fermentation time was examined and shown to impact solute levels. In addition, the drying regime was varied to produce samples dried to different moisture contents. The experiments show greater drying led to higher mannitol levels, while trehalose levels remained constant. Rapid drying of the sclerotia in a fluidized-bed dryer also show rapid accumulation of mannitol and trehalose, which suggest the enzyme activities needed for production are readily available. The findings confirm mannitol and trehalose are important metabolites in M. terrestris and their concentrations are responsive to osmotic stress conditions.     

金针菇菌种中短期保藏因素的正交分析

【背景】金针菇(Flammulina velutipes)是我国一种重要的栽培食用菌,年产量超过250万t,规模已跃居世界首位。菌种保藏技术是金针菇栽培和新品种研发的基础,但相关研究十分薄弱,已成为制约我国金针菇产业进一步发展的瓶颈问题。【目的】探索不同保藏因素对金针菇优良菌种中短期保藏的影响,为建立高效、低成本、易操作的保藏方法奠定基础。【方法】以温度、甘油、海藻糖、甘露醇以及保护剂体积5个因素进行正交试验。经12个月保藏,考察金针菇菌种在木屑培养基中的菌丝生长速度,通过极差分析和回归分析解析保藏因素的效应。【结果】温度、海藻糖、甘油和甘露醇对金针菇菌种的中短期保藏有极显著的影响,保护剂体积的影响不显著。温度是最重要的影响因子,与其它4个因素的互作效应均达到极显著水平。20°C是较好的短期保藏温度,-80°C为理想的中期保藏温度。渗透型与非渗透型保护剂间的互作效应对金针菇菌种的中短期保藏有极显著影响,海藻糖和甘露醇间的互作效应不显著。高浓度的海藻糖、甘油及甘露醇均不利于金针菇菌种的中短期保藏。保藏效果较佳的保护剂为10%甘油和0.3 mol/L甘露醇混合液。【结论】建立的菌种中短期保藏方法填补了金针菇产业发展的空白,研究结果可为其它大型真菌的中短期保藏提供重要参考。     

Carbohydrates in the hypothallus and areolae of the crustose lichen <Emphasis Type="Italic">Rhizocarpon geographicum</Emphasis> (L.) DC.

Carbohydrate concentrations in the marginal hypothallus and areolae of the crustose lichen Rhizocarpon geographicum (L.) DC. were measured in north Wales, U.K. using gas chromatography. Ribitol, arabitol, and mannitol were the most abundant carbohydrates while α- glucose β-glucose, fructose, sucrose, and trehalose were present in smaller amounts. The concentrations of arabitol, ribitol, mannitol, fructose, and α-glucose were greater in the areolae while the concentration of trehalose was greater in the hypothallus. Concentrations of carbohydrates varied little between sample days. Concentrations of polyols in the hypothallus were not correlated with those in the areolae. These results suggest: 1) the hypothallus has a lower demand for carbohydrates than the areolae or there is limited transport from areolae to hypothallus, 2) increased trehalose in the non-lichenised hypothallus may be an adaptation to withstand stress and desiccation, and 3) polyols are partitioned differently in the hypothallus and areolae.     

Physiological implication of intracellular trehalose and mannitol changes in response of entomopathogenic fungus <Emphasis Type="Italic">Beauveria bassiana</Emphasis> to thermal stress

To explore possible role of intracellular trehalose accumulation in fungal tolerance to summer-like thermal stress, 3-day colonies of Beauveria bassiana grown on a glucose-free medium at 25°C were separately exposed to 35, 37.5 and 40°C for 1–18 h, respectively. Trehalose accumulation in stressed mycelia increased from initial 4.2 to 88.3, 74.7 and 65.5 mg g⁻¹ biomass after 6-h stress at 35, 37.5 and 40°C, respectively, while intracellular mannitol level generally declined with higher temperatures and longer stress time. The stress-enhanced trehalose level was significantly correlated to decreased trehalase activity (r ² = 0.73) and mannitol content (r ² = 0.38), which was inversely correlated to the activity of mannitol dehydrogenase (r ² = 0.41) or mannitol 1-phosphate dehydrogenase (r ² = 0.30) under the stresses. All stressed cultures were successfully recovered at 25°C but their vigor depended on stressful temperature, time length and the interaction of both (r ² = 0.98). The highest level of 6-h trehalose accumulation at 35°C was found enhancing the tolerance of the stressed cultures to the greater stress of 48°C. The results suggest that the trehalose accumulation result partially from metabolized mannitol and contribute to the fungal thermotolerance. Trehalase also contributed to the thermotolerance by hydrolyzing accumulated trehalose under the conditions of thermal stress and recovery.