Natural resistance of the mycelial culture of the mushroom,Panaeolus papillonaceus,towards growth inhibition by polyene antibiotics

The mycelial culture of the mushroomPanaeolus papillonaceus showed high tolerance (150 g/ml) of polyene antibiotics (nystatin, amphotercinin B) present in the growth medium and protoplast of the fungus regenerated normally in the presence of the antibiotics. Both antibiotics inhibited growth of other mushroom strains at concentrations from 10 g/ml to 20 g/ml. Because polyene antibiotics interact with free membrane sterol of the sensitive fungi, the sterol present in the mycelia ofP. papillonaceus was studied. Extraction of sterol from the mushroomP. papillonaceus required primary treatment of the dried mycelia with alkali, and only ergosterol was identified as present as the extracted sterol. No sterol or sterol conjugate (fatty acid ester) could be extracted directly from the mycelia by petroleum ether, chloroform, or methanol without prior alkali treatment. Homogenization of the mycelia and subsequent treatment of the homogenate with detergent or chaotropic ions did not release any sterol conjugate in the aqueous phase. The unique nature of the sterol component present in the mycelia ofP. papillonaceus was indicated.     

Evolutionary relationships of membrers of the generaTaphrina, Protomyces, Schizosaccharomyces, and related taxa within the archiascomycetes: Integrated analysis of genotypic and phenotypic characters

To study the phylogeny and evolution of archiascomycetes, we determined the full sequence of the nuclear 18S rRNA gene from 14Taphrina species and 2Protomyces species, and the partial sequence ofSchizosaccharomyces japonicus var.japonicus. The sequences were phylogenetically analyzed by the neighbor-joining, maximum parsimony, and maximum-likelihood methods. We also looked at their principal phenotypic characters and genotypic character. Relationships within the Ascomycota are concordant with the previously published phylogenies inferred from 18S rDNA sequence divergence and divide the archi-, hemi-and euascomycetes into distinct major lineages. All the trees show that, within the archiascomycete lineage, 11 of the 14Taphrina species and the 2Protomyces species are monophyletic. A core groups ofTaphrina andProtomyces is always monophyletic. The evidence from molecular and phenotypic characters such as cell wall sugar composition, ubiquinone, cell wall ultrastructure, and mode of conidium ontogeny, strongly suggests that ‘T’. californica CBS 374.39, ‘T’. maculans CBS 427.69 and ‘T’. farlowii CBS 376.39 should be excluded from the archiascomycete lineage. ‘Taphrina’ farlowii CBS 376.39 groups withCandida albicans in the Saccharomycetales, whereas ‘T’. californica CBS 374.39 and ‘T’. maculans CBS 427.69 have a basidiomycete affinity and group with Tremellalean members in the hymenomycete lineage.Schizosaccharomyces is monophyletic. The strictly anamorphic yeastSaitoella complicata groups with the apothecial ascomyceteNeolecta vitellina rather than theTaphrina/Protomyces branch.     

The extraction and quantification of ergosterol from ectomycorrhizal fungi and roots

Recently, ergosterol analysis has been used to quantify viable fungal biomass in resynthesized ectomycorrhizae. An objective of our study was to quantify ergosterol in a range of ectomycorrhizal isolates under differing growth conditions. In addition, we tested the applicability of the method on field-collected roots of ectomycorrhizal and vesicular-arbuscular (VA) mycorrhizal plants. Quantification of sitosterol as a biomass indicator of plant roots was also undertaken. Ergosterol was not detected in roots of uninoculated Betula populifolia seedlings, and sitosterol was not detected in an ectomycorrhizal fungal isolate but was present in birch roots. Ergosterol was produced in all isolates examined, which represented the major orders of ectomycorrhizal fungi. The range of values obtained, from 3 to nearly 18 g ergosterol mg^-1 dry mass, agrees well with reported values for other mycorrhizal and decomposer fungi. Hyphal ergosterol was the same during growth on phytic acid and KH₂PO₄. Reduction of growth temperature from 25° C to 15° C had little effect on ergosterol content of cultures harvested at similar growth stages. Ergosterol and sitosterol were detected in field-collected ectomycorrhizae of B. populifolia and Pinus sylvestris and VA mycorrhizae of Acer rubrum and Plantago major. Both ergosterol content and ergosterol to sitosterol ratios were significantly lower in VA mycorrhizae than ectomycorrhizae. Calculations of viable fungal biomass associated with field-collected roots were in agreement with those reported by others using the method on resynthesized ectomycorrhizae. Estimates of total mass could be obtained for field-collected B. populifolia roots by a simultaneously using ergosterol to estimate fungal biomass and sitosterol to estimate root mass. Some potential applications and limitations of sterol quantification in studies of mycorrhizal physiology and ecology are discussed.     

The effects of Tunicamycin and 2-deoxy-D-glucose on the development ofXenopus laevis embryos

Summary Tunicamycin and 2-deoxy-D-glucose were applied toXenopus laevis embryos in the first cleavage furrow, blastula and early gastrula stages. No effect was observed with 2-deoxy-D-glucose up to the concentration 0.1 M. The effect of Tunicamycin is dose- and stage-dependent. At the concentration of 5 g/ml cleaving embryos are arrested at the onset of gastrulation and their cells exhibit decreased intercellular adhesivity, while embryos treated in the blastula and early gastrula stages may develop up to the late neurula and tail-bud stage, respectively. Higher concentrations (up to 20 g/ml) drastically affect cleavage. Concentrations of 4 to 1 g/ml allow embryos to develop up to more advanced stages; however, developmental defects are the rule. Concentrations of less than 1 g/ml do not affect development.     

Sterols of Goniotrichum elegans

The sterol fraction was examined from cultures of the filamentous red alga Goniotrichum elegans. Brassicasterol accounted for nearly half of the total sterol and was accompanied by cholesterol (24%) as well as a number of minor components. This is the first record of brassicasterol as a major sterol in the Rhodophycophyta. The occurrence of this C₂₈ major sterol may be of taxonomic importance in determining the relationship of the Goniotrichales to the other red algae of which have C₂₇ major sterols.     

Rapid distinction between micrococci and staphylococci with furazolidone agars

Furazolidone agar proved to be a suitable medium for separating strains of the genera Micrococcus and Staphylococcus. 720 isolates (including 24 type strains) of gram- and catalase-positive cocci were tested for growth on tryptone soya and peptone agar with the addition of 50 g/ml furazolidone. The results were compared with the classification obtained by the standard-O/F-test and by the test system of Schleifer and Kloos.For routine identification and separation of staphylococci from micrococci a peptone agar with 20 g furazolidone/ml is recommended.     

Biotransformation of mercury by bacteria isolated from a river collecting cinnabar mine waters

One hundred six strains of aerobic bacteria were isolated from the Fiora River which drains an area of cinnabar deposits in southern Tuscany, Italy. Thirty-seven of the strains grew on an agar medium containing 10g/ml Hg (as HgCl₂) with all of these strains producing elemental mercury. Seven of the 37 strains also degraded methylmercury. None of 106 sensitive and resistant strains produced detectable monomethylmercury although 15 strains produced a benzene-soluble mercury species. Two strains of alkylmercury (methyl-, ethyl- and phenylmercury) degrading bacteria were tested for the ability to degrade several other analogous organometals and organic compounds, but no activity was detected toward these compounds. Mercury methylation is not a mechanism of Hg resistance in aerobic bacteria from this environment. Growth of bacteria on the agar medium containing 10g/ml HgCl₂ was diagnostic for Hg detoxification based on reduction.     

Utilization of cheese whey for lipid and sterol production by some isolates of xerophilous fungi

One hundred and two isolates of 30 species and 4 varieties belonging to 4 genera of xerophilous fungi were cultivated on cheese whey. In fourteen species and one variety, all isolates (45) failed to grow on the cheese whey. The other species and varieties (16 and 3) of fungi were able to grow and to accumulate variable amounts of lipid and sterol on whey.Aspergillus aureolatus, Eurotium repens, E. rubrum andEmericella nidulans var.lata produced high levels of lipids, total sterols and ergosterol whileE. tonophilum proved to be superior in the production of lipid (38±8%) of dry mycelial mass), total sterol (20±4%) and ergosterol (13±3%).     

Ergosterol content of Rhizopus oligosporus NRRL 5905 grown in liquid and solid substrates

Summary The ergosterol content of Rhizopus oligosporus NRRL 5905 varied between 2–24 g/ mg biomass dry matter when grown in laboratory media and was found to be influenced by the substrate composition.When grown on a natural substrate (soya beans) the ergosterol content was considerably higher (estimated at approx. 60–90 g/mg biomass dry matter).In laboratory media, the ergosterol content was also influenced by the extent of aeration and the growth phase of the mycelium; within the range of 25° C–35° C, the incubation temperature did not influence the ergosterol content significantly.In view of these variations, ergosterol should not be used as a chemical index for the quantification of biomass grown in static solid-substrate fermentations with limited mass transfer, e.g. tempe or oncom.     

Fungal biomass and decomposition in Spartina maritima leaves in the Mondego salt marsh (Portugal)

Spartina maritima (Curtis) Fernald is a dominant species in the Mondego salt marsh on the western coast of Portugal, and it plays a significant role in estuarine productivity. In this work, leaf litter production dynamics and fungal importance for leaf decomposition processes in Spartina maritima were studied. Leaf fall was highly seasonal, being significantly higher during dry months. It ranged from 42 g m^-2 in June to less than 6 g m^-2 during the winter. Fungal biomass, measured as ergosterol content, did not differ significantly between standing-decaying leaves and naturally detached leaves. Fungal biomass increased in wet months, with a maximum of 614 g g^-1 of ergosterol in January in standing-decaying leaves, and 1077 g g^-1 in December, in naturally detached leaves, decreasing greatly in summer. Seasonal pattern of fungal colonization was similar in leaves placed in litterbags on the marsh-sediment surface. However, ergosterol concentrations associated with standing-decaying and naturally detached leaves were always much higher than in litterbagged leaves, suggesting that fungal activity was more important before leaf fall. Dry mass of litterbagged leaves declined rapidly after 1 month (about 50%), mostly due to leaching of soluble organic compounds. After 13 months, Spartina leaves had lost 88% of their original dry weight. The decomposition rate constant (k) for Spartina maritima leaves was 0.151 month^-1.     

Sterol content and enzyme defects of nystatin-resistant mutants of Neurospora crassa.

Summary Non-saponifiable cell extracts of wild type and sterol mutants of N. crassa were analysed by means of gas-liquid chromatography. The wild-type contained ergosterol and episterol in a 10:1 ratio. None of the mutants was able to synthesize ergosterol. Three of the mutants carry single recessive gene mutations causing blocks in the terminal steps of ergosterol biosynthesis: erg-1 has an inactive ^{8 7} isomerase, erg-2 has an inactive 24(28) hydrogenase, and erg-4 has an inactive C-24 methyl transferase. Some of the mutants accumulated novel sterols as a result of their enzyme defects. The genes erg-1 and erg-2 were mapped close to inl on the right arm of chromosome V.     

Determination of the minimal inhibitory concentration of Amphotericin B and Miconazole for 21 strains of Aspergillus

The susceptibility of 21 strains ofAspergillus (11 ofA. fumigatus, 8 ofA. niger, and 2 ofA. flavus) isolated from human pathologic specimens to Amphotericin B and Miconazole has been comparatively studied. Determination of the minimal inhibitory concentration of both drugs in a liquid medium showed a noticeably variability for the different strains. The values obtained for Amphotericin B varied between 0.25g/ml (2 strains) and 1.25g/ml (5 strains) after 48 hours, and between 1.25g/ml (1 strain) and 50g/ml (1 strain) after 10 days. For Miconazole the results varied between 0.1g/ml (1 strain) and 25g/ml (1 strain) after 48 hours of incubation, and between 0.5g/ml (5 strains) and > 100g/ml after 10 days. The variability of these results indicates the usefulness of carrying ourin vitro sensitivity studies whenever it is possible.     

Effects of Ginkgo Biloba Extract Constituents on Glycine-Activated Strychnine-Sensitive Receptors in Hippocampal Pyramidal Neurons of the Rat

Active compounds of Ginkgo biloba extract (ginkgolides A, B, C, and J) were tested on the main ligand-operated conductances in rat neurons using a patch-clamp technique in the whole-cell configuration and a concentration-clamp technique. It was found that all ginkgolides reduced glycine-activated currents in concentration- and use-dependent manners, whereas they did not affect other tested ligand-gated receptors (NMDA- and GABA-activated ones). The IC₅₀ values calculated from dose-response curves were as follows: 1.97, 0.273, 0.267, and 2.0 M for ginkgolides A, B, C, and J, respectively (200 M Gly ).The Hill coefficient in all cases was close to 1.0, which indicates a single site of the drug binding to the glycine-activated receptor.     

Extraction of sterols in brown macroalgae from Antarctica and their identification by liquid chromatography coupled with tandem mass spectrometry

This study reports for the first time the extraction and quantification of sterols in six species of brown macroalgae from Antarctica: Adenocystis utricularis, Ascoseira mirabilis, Cystosphaera jacquinotii, Desmarestia anceps, Desmarestia antarctica, and Himantothallus grandifolius. Ultrasound irradiation was used as a promotor to extract sterols from algal biomass. The extracts were analyzed by high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for sterol quantization (ergosterol, brassicasterol, fucosterol, β-sitosterol, campesterol, cholesterol, and stigmasterol). In general, fucosterol was the most abundant (6.60 to 48.13 mg kg^?1), followed by β-sitosterol (5.29 to 16.49 mg kg^?1), stigmasterol (2.69 to 14.84 mg kg^?1) in five of the six examined algae. The sterol campesterol was found in lower concentrations (0.07 to 0.15 mg kg^?1) in all studied samples.     

The production of azurin and similar proteins

Summary It has been shown that, in the organisms tested, the production of azurin or similar blue, copper-protein complexes is confined to bacterial species of the three genera Bordetella, Alcaligenes and Pseudomonas. In the strain of Pseudomonas aeruginosa used, there appeared to be no difference in the amount of azurin occurring when the strain was grown aerobically and anaerobically. The amount of azurin produced by representative strains of Ps. aeruginosa, B. bronchiseptica and A. denitrificans varied with the copper content of the medium. Above a level of 5 g copper/ml of medium, the azurin content was constant for the three species tested; below a copper level of 0.5 g/ml there was an almost total absence of azurin although good growth occurred. Under similar growth conditions, the azurin content of the three bacterial species studied was not significantly different.The possible role of azurin is discussed.     

The composition of Erythrosins, Fluorescein, Phloxine and Rose Bengal: a study using thin-layer chromatography and solvent extraction

Synopsis Commercial samples of Erythrosin B (CI 45430), Erythrosin Y (CI 45425), Fluorescein (CI 45350), Phloxine (CI 45410) and Rose Bengal (CI 45440) have been analysed by thin-layer chromatography. The Erythrosins were found to be mixtures consisting in the main of 4-iodofluorescein, 4,5-di-iodofluorescein, 2,4,5-triiodofluorescein and 2,4,5,7-tetraiodofluorescein, in some instances together with 2,4,5-tri-iodo-4,5,6,7-tetrachlorofluorescein and 2,4,5,7-tetraiodo-4,5,6,7-tetrachlorofluorescein. Samples of Fluorescein were mixtures of the nominal dye usually with traces of several unidentified, fluorescent components. Those of Phloxine consisted mainly of mixtures of 4-bromo-4,5,6,7-tetrachlorofluorescein, 4,5-dibromo-4,5,6,7-tetrachlorofluorescein, 2,4,5-tribromo-4,5,6,7-tetrachlorofluorescein and 2,4,5,7-tetrabromo-4,5,6,7-tetrachlorofluorescein, often with 4,5,6,7-tetrachlorofluorescein Samples of Rose Bengal were mixtures of 4-iodo-4,5,6,7-tetrachlorofluorescein, 4,5-di-iodo-4,5,6,7-tetrachlorofluorescein, 2,4,5-tri-iodo-4,5,6,7-tetrachlorofluorescein and 2,4,5,7-tetraiodo-4,5,6,7-tetrachlorofluorescein together with some unidentified components.Most of the commercial dye samples gave an insoluble residue when extracted with methanol. This residue was usually inorganic carbonate or halide. Some possible practical consequences of the various impurities are discussed.     

Characteristics of 0/129-sensitive motile Aeromonas strains isolated from freshwater on starch-ampicillin agar

Motile Aeromonas hydrophila strains were recovered from several freshwater sources by spread-plating water samples on starch-ampicillin agar, originally described as a medium for recovering Aeromonas hydrophila quantitatively from foods. Starch-ampicillin agar was compared with membrane Aeromonas medium and Rimler-Shotts medium for selectivity for, and recovery of, Aeromonas strains from freshwater. Thirty-four Aeromonas strains thus isolated were identified to species level by their phenotypic characteristics, and the Mol% G+C of representative strains was determined. Although resistant to 10 g of the vibriostatic agent 0/129, all these strains showed sensitivity to 150 g 0/129, which brings into question the use of this agent for distinguishing aeromonads from vibrios. The ability of these strains to produce extracellular virulence factors was generally similar to that reported for environmental strains isolated by other methods from various geographical locations within and beyond Australia. Ten of the 20 A. sobria strains, but none of the A. hydrophila or A. caviae strains, produced enterotoxin as shown by the suckling mouse test. Haemolysin was produced by 9/10 of the enterotoxigenic A. sobria strains and 2/9 A. hydrophila strains. Hemagglutinating activity was detected in 5/20 A. sobria and 7/9 A. hydrophila strains, and was inhibited by fucose and mannose, but not by galactose. The characteristics of these strains were comparable with those of Aeromonas strains isolated from other freshwater environments apart from their sensitivity to 0/129. Send offprint requests to: M. Cahill.     

Search for A-factor-Dependent Variants in Actinomycete Populations

A study of 28 nocardia-like, asporogenous, and oligosporous spontaneous morphological variants belonging to 23 species of streptomycetes revealed five strains producing regulators of the A-factor group. Streptomyces griseus 1439, which forms aerial mycelium and spores only in the presence of exogenous A-factor, was used as the test strain. Among the 28 spontaneous variants, three new A-factor-dependent strains were revealed, which represented the species Streptomyces griseus, S. citreofluorescens, and S. viridovulgaris subsp. albomarinus. These weakly differentiated variants did not produce A-factor and behaved as its recipients, responding by changes in their morphological characteristics at a concentration of this regulator in the medium of 0.01 g/ml or higher. The original collection strains in whose populations the variants were selected produced substances of the A-factor group. The A-factor-dependent variants differed in the level of the regulator required for maximal expression of the morphological characteristics: it was necessary to introduce the A-factor at a concentration of 1 g/ml for S. citreofluorescens and S. viridovulgaris subsp. albomarinus and at 10 g/ml for S. griseus.     

SelectingRhizobium phaseoli strains for use with beans (Phaseolus vulgaris L.) in Kenya: Infectiveness and tolerance of acidity and aluminium

Forty strains ofRhizobium phaseoli, isolated from Kenyan soils, were tested for infectiveness on common bean (Phaseolus vulgaris L.). 28 strains were infective and a cultivar × Rhizobium interaction was observed. 48 strains were screened for tolerance of acidity and Al in liquid culture. Assessment of visible turbidity after 14 days indicated 34 strains tolerant of pH 4.5 but none tolerant of pH 3.5. No strain was tolerant of 50 M Al at pH 5.5. Three strains were tolerant of 20 M Al at pH 5.5 and 10 M Al at pH 4.5. Screening on a solid medium identified strains tolerant of 20 and 50 M Al at pH 5.5 and 4.5 which were sensitive to these treatments in liquid culture. Those strains tolerant to 20 M Al at pH 4.5 and 5.5 in liquid culture were correctly identified on the solid medium.     

Carotenoid Pigments of some Lower Ascomycetes

Protomyces pachydermus and P. inouyei contains ß-,-carotene and lycopene, with ß-carotene predominating.Taphrina deformans and T. communis contains no carotenoid pigmentsalthough the colour of methanol extracts of all four speciesis yellow. A quick way of differentiating between these twogenera is by showing the presence of carotenoid pigments inProtomyces and absence of these in Taphrina. It is suggestedthat a knowledge of individual carotenoid pigments in fungimight be useful as a taxonomic tool among fairly closely relatedspecies.