Distribution of carotenoids and sterols in relation to the taxonomy of Taphrina and Protomyces

Species of the genera Taphrina Fr. and Protomyces Unger were screened for the presence of carotenoid pigments and the sterols ergosterol and brassicasterol. All strains produced carotenoids in variable amounts: Taphrina: 0.3–39 g/g dry weight; Protomyces: 65–99 g/g dry weight. It was concluded that the tow genera cannot be separated on the basis of presence or absence of carotenoids. Thirty strains (24 species) of Taphrina produced brassicasterol as the principal sterol; twenty-one strains (17 species) did not form ergosterol. Only four isolates (4 species) produced ergosterol without formation of brassicasterol. Brassicasterol was the major sterol in 3 species of Protomyces, whereas ergosterol was absent. Brassicasterol is a rather unique sterol within the fungal kingdom and has hitherto not been found in the red yeasts. Therefore, this sterol is of taxonomic significance in contrast with ergosterol, which is widespread among fungi.     

Fungi on Leaf Blades of <Emphasis Type="Italic">Phragmites australis</Emphasis> in a Brackish Tidal Marsh: Diversity,Succession, and Leaf Decomposition

Although fungi are known to colonize and decompose plant tissues in various environments, there is scanty information on fungal communities on wetland plants, their relation to microhabitat conditions, and their link to plant litter decomposition. We examined fungal diversity and succession on Phragmites australis leaves both attached to standing shoots and decaying in the litter layer of a brackish tidal marsh. Additionally, we followed changes in fungal biomass (ergosterol), leaf nitrogen dynamics, and litter mass loss on the sediment surface of the marsh. Thirty-five fungal taxa were recorded by direct observation of sporulation structures. Detrended correspondence analysis and cluster analysis revealed distinct communities of fungi sporulating in the three microhabitats examined (middle canopy, top canopy, and litter layer), and indicator species analysis identified a total of seven taxa characteristic of the identified subcommunities. High fungal biomass developed in decaying leaf blades attached to standing shoots, with a maximum ergosterol concentration of 548 ± 83 μg g^–1 ash-free dry mass (AFDM; mean ± SD). When dead leaves were incorporated in the litter layer on the marsh surface, fungi experienced a sharp decline in biomass (to 191 ± 60 μg ergosterol g^–1 AFDM) and in the number of sporulation structures. Following a lag phase, species not previously detected began to sporulate. Leaves placed in litter bags on the sediment surface lost 50% of their initial AFDM within 7 months (k = −0.0035 day^–1) and only 21% of the original AFDM was left after 11 months. Fungal biomass accounted for up to 34 ± 7% of the total N in dead leaf blades on standing shoots, but to only 10 ± 4% in the litter layer. These data suggest that fungi are instrumental in N retention and leaf mass loss during leaf senescence and early aerial decay. However, during decomposition on the marsh surface, the importance of living fungal mass appears to diminish, particularly in N retention, although a significant fraction of total detrital N may remain associated with dead hyphae.     

Regulation of sterol biosynthesis inSaccharomyces cerevisiae

Sterol synthesis inSaccharomyces cerevisiae was primarily controlled by the growth rate. At low specific growth rates the intermediates of ergosterol biosynthesis prevailed in cells. At the same time, the total sterol content reached about 6% of dry matter whereas the content of ergosterol was only 2–2.5%, which seems to be the maximum value forS. cerevisiae. After esterification with fatty acids these sterol intermediates are stored in lipid globules together with reserve triacylglycerols. The sporulatingS. cerevisiae cells contained 3.5% sterols and 1.5% ergosterol of dry matter.     

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.     

Seasonal variation of fungal biomass in the sediment of a salt marsh in New Brunswick

In a marsh in New Brunswick, Canada, belowground biomass of Spartina alterniflora consistently exceeded aboveground biomass by a factor of approximately 9. Both values peaked in July. Redox potential of the sediment was negative at all levels tested (2, 6, and 11 cm below surface), and was negatively correlated with depth. Concentrations of ergosterol, a sterol typical of higher fungi, were negatively correlated with redox potential and were highest in roots and rhizomes in July and August, 1–3 cm below the surface. These maxima corresponded to a fungal content of approximately 0.6% per ash-free dry mass of Spartina material. Balsa wood panels buried in anaerobic salt marsh sediment were colonized by fungi within 12 weeks. Eight fungal species isolated from S. alterniflora roots did not grow in the absence of oxygen, but were able to grow downward into an anaerobic medium. Offprint requests to: F. Bärlocher.     

Incidence of seed-borne fungi and aflatoxins in Sudanese lentil seeds

Thirteen seed samples of lentil (Lens esculenta) were incubated on agar plate and moist filter papers (Moist Chambers) at 28 ± 2 °C for determination of the incidence of seed-borne fungi. Aflatoxins content of the seeds was measured using the bright greenish-yellow fluorescence test (BGYF) and thin-layer chromatography (TLC). Sixty-nine species and seven varieties, which belong to 24 genera of fungi, were isolated from this crop. Of these fungi, 51 species and two varieties are considered new for this crop, whereas seven genera and 13 species are new to the mycoflora of the Sudan. The genus Aspergillus (13 species and 6 varieties) which comprising 44% of the total colony count was the most prevalent genus followed by Rhizopus (2 species, 19%), Penicillium (6 species) and Fusarium (8 species) (12%), Chaetomium (3 species) and Cladosporium (5 species) (6%), where the 18 genera (1–4 species) showed very low level of incidence (19%). Of the possible pathogens of lentil plants, F. oxysporum the main cause of vascular wilt was recovered from seeds of this crop. Thin layer chromatographic analysis of chloroform extracts of 13 seed samples showed that only one samples was naturally contaminated with aflatoxins B₁, B₂, G₁ and G₂ (14.3 μg/kg). This revised version was published online in June 2006 with corrections to the Cover Date.     

Physiological characteristics of 13 common fungal species in bathrooms

To clarify the factors affecting fungal contamination in bathrooms, the growth of 13 common fungal species (13 isolates) in bathrooms was studied under various environmental conditions. Most of the fungi examined grew on media of 0.01% and 0.05% sodium fatty acid and on media of 0.01% anion surfactant. On media of non-ion surfactant, however, growth varied from species to species. Fungi found commonly in bathrooms can be divided into two groups. The first group, including six species — Cladophialophora boppii, Exophiala spinifera, E. salmonis, Phialophora europaea, Phoma herbarum, and Scolecobasidium constrictum — grew on media of 0.01%, 0.05%, and 0.25% non-ion surfactant, with the latter five species also growing on alkali medium. Most of them did not grow at 33°C, or on media with 10% NaCl, however. Fungi of these six species, identified using DNA and morphological analysis, were common in bathrooms, but not in other indoor environments, for example, in house dust or on windows. The second group contained seven species including Aureobasidium sp., Cladosporium cladosporioides, and Fusarium sp., which were common both in house dust and in bathrooms; they did not grow on media of 0.05% or 0.25% non-ion surfactant, but most grew comparatively fast on normal medium (1/4 PDA), and were able to grow on media with 10% NaCl and also at 33°C. The characteristic fungi found in bathrooms were able to exploit surfactant but were unable to grow well under comparatively dry or high-temperature conditions.     

Sterols synthesized by cultured trichomycetes

Desmosterol was synthesized by all but 2 of 14 isolates of the fungal genus Smittium (Harpellales) obtained from the hindguts of various Diptera larvae and grown axenically on a sterol-free medium. Also detected in some isolates of Smittium were cholesterol and ergosterol, and another, unidentified sterol with an apparent molecular ion of 470. Sterol content based on dry weight of fungal tissue was as high as 0.34%, but it was shown in one isolate that sterols vary both quantitatively and qualitatively during growth of the fungus. Two axenic cultures of the trichomycete Amoebidium parasiticum (Amoebidiales) isolated from the exoskeleton of a Cladocera contained cholesterol and ergosterol, but no desmosterol. Two soil fungi with possible phylogenetic affinities to Smittium, Linderina pennispora and Dipsacomyces acuminosporus (Kickxellales), produced cholesterol alone. The possible influence of the gut fungi on arthropod growth and development is discussed.Abbreviations TMS trimethylsilyl Based upon a Master of Arts thesis submitted by the senior author to the Department of Botany, University of Kansas. This research was partially supported by NSF grants BMS 72-02380-AO and DEB 77-16161 to R.W.L.     

Effect of the high polycyclic aromatic hydrocarbon, benzo[a]pyrene, on the lipid content of Fusarium solani

The purpose of the present paper was to study the effect of the high polycyclic aromatic hydrocarbon (PAH), benzo[a]pyrene, on the lipid [fatty acid (FA) and sterol] composition and content of the fungi Fusarium solani and F. oxysporum, respectively recognized as good and poor PAH degraders. The major FAs and the major sterol that characterized the tested Fusarium strains were C16:0, C18:1, C18:2, and ergosterol. Lipid profiles of F. solani remained unchanged with the addition of benzo[a]pyrene in the culture media at all concentrations and duration of treatment. However, in the presence of benzo[a]pyrene, significant decreases in FA content, which reached 18 % in young cultures and 28 % in mature colonies, were registered. Similarly, the sterol content of F. solani was reduced by 27 % in the presence of benzo[a]pyrene. In contrast, no modification in lipid profile and lipid content were observed with F. oxysporum, a strain recognized as a low benzo[a]pyrene degrader.     

Antimicrobial activity of resin acid derivatives

The wide potential of resin acids as bioactive agents gave rise to a growing effort in the search for new applications of the natural forms and their derivatives. In some of these compounds, the antimicrobial activity is associated to the presence in the molecules of functional groups such as the hydroxyl, aldehyde, and ketone or to their cis or trans configurations. The resin acid family covers a spectrum of antimicrobial activities against several microorganisms, from bacteria to fungi, in which the mode of action was studied by electron microscopy. The morphological alterations are consistent with an unspecific mode of action causing inhibition of the fungal growth or damaging the fungal cells in parallel with a mechanism of resistance based on the retention of the compound by the lipid accumulation. The sterol composition of phytopathogenic fungi Botrytis cinerea and Lophodermium seditiosum treated with methyl cis-7-oxo-deisopropyldehydroabietate revealed the presence of ergosterol (M+ 396) and dihydroergosterol (M+ 398) in both cultures showing that this compound did not interfere with the ergosterol metabolic pathway of both fungi.     

Effect of Membrane Structure on the Action of Polyenes II: Nystatin Activity along the Phase Diagram of Ergosterol- and Cholesterol-Containing POPC Membranes

Pores formed by the polyene antibiotic nystatin were studied in solvent-free lipid membranes. The membranes were formed by the tip-dip technique using 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) with different mol fractions (0–50%) of cholesterol or ergosterol. The effects of the mol fraction of sterol and of temperature variation (15–35°C) on the activity of the pores, their unitary conductances, lifetimes and time average conductances were studied. The results were used to analyze the behavior of nystatin channels along the phase diagrams previously reported for these lipid mixtures and to propose that membrane structure is the determinant factor for the known ergosterol/cholesterol selectivity.     

Antibacterial Effect of Bovine Lactoferrin Against Udder Pathogens

The antibacterial effect of lactoferrin (Lf) was tested on isolates of Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and coagulase-negative staphylococci (CNS) as well as on Pseudomonas aeruginosa (P. aeruginosa) and Klebsiella pneumoniae (K. pneumoniae), originally isolated from bovine mastitis. Concentrations of Lf used were 0.67 mg/ml, 1.67 mg/ml, and 2.67 mg/ml. Growth of udder pathogens was monitored by turbidometry either in broth culture or in whey prepared from normal milk. We focused on 3 different growth variables: lag time, slope, and maximum absorbance of bacterial growth curves. Growth inhibition was seen in the broth but hardly at all in whey. The isolates of E. coli and CNS did not grow sufficiently well in whey to draw any conclusions. The most effective inhibitory activity of Lf was seen against E. coli and P. aeruginosa. All 5 E. coli isolates had similar growth patterns. Inhibition of growth by Lf was concentration-dependent. The concentration of 0.67 mg/ml in broth and whey was generally too low for a significant inhibitory effect.     

Seasonal ectomycorrhizal fungal biomass development on loblolly pine (Pinus taeda L.) seedlings

Ergosterol, a membrane sterol found in fungi but not in plants, was used to estimate live mycelial biomass in ectomycorrhizae. Loblolly pine (Pinus taeda L.) seeds were sown in April 1993 and grown with standard nursery culture practices. Correlations between total seedling ergosterol and visual assessment of mycorrhizal colonization were high during July and August but low as ectomycorrhizal development continued into the growing season. Percentages of mycelial dry weight over lateral roots decreased from 9% in July to 2.5% in November because seedling lateral root dry weight accumulated faster than mycelial dry weight. Total ergosterol per seedling increased from July through February. As lateral root dry weight ceased to increase during winter months, ectomycorrhizal mycelia became the major carbohydrate sink of pine seedlings. No distinctive seasonal pattern of soil ergosterol content was observed. The impact of ectomycorrhizal fungi on plant carbohydrate source-sink dynamics can be quantitatively estimated with ergosterol analysis but not with conventional visual determination.     

The effect of the preservative sorbic acid on the lipid composition of the ascomycete fungus <Emphasis Type="Italic">Penicillium roqueforti</Emphasis> Thom

The mechanism of action of potassium sorbate, a widely used food preservative on the lipid composition of the Ascomycete fungus Penicillium roqueforti, the main contaminant of cheese, was investigated. The inhibition of fungal growth by potassium sorbate was found to be associated with a change in the composition of phospholipids (a decrease in phosphatidylcholine content and an increase in phosphatidylethanolamine and phosphatidic acid content) and of neutral lipids (a decrease in the triacylglycerol and sterol content and an increase in the free fatty acid content). The fatty acid composition of fungal lipids also changed. A drastic decrease in the linoleic acid content occurred both in the total lipid fraction and in the triacylglycerol and total phospholipid fractions, whereas the oleic acid content increased correspondingly. This suggests that sorbic acid (SA) affects Δ12 desaturase activity, which controls the adaptive response of mycelial fungi to deleterious environmental factors.     

Microbial flora of some soils of Mawson Base and the Vestfold Hills,Antarctica

Summary A microbiological survey of 40 soils from contaminated and pristine localities of Mawson Base or the Vestfold Hills (Davis Base) is described. High microbial populations (from 10⁶ to in excess of 10⁹ cells g^-1 dry soil) were associated with soil contaminated by man or animals, soil from beneath a moss bed, or soil from beneath translucent quartz pebbles and containing green algae. Lower microbial populations (generally between 10⁴–10⁶ cells g^-1 soil) were associated with pristine soils lacking an identifiable primary producer. No correlation was evident between population density and soil particle size, moisture content, pH or salinity as determined by the electrical conductivity (E.C.) of soil suspensions, although relationships were evident between the components of the microflora and E.C. Microbial groups associated with low E.C. were chitinolytic bacteria, fungi and green algae; favoured by high E.C. were halotolerant bacteria. The microflora of uncontaminated soils appeared to be dominated by Gram-negative aerobic rods (68% of the total flora) with Moraxella, Acinetobacter and Pseudomonas being the most frequently isolated genera. The remaining microflora comprised Grampositive pleomorphic bacteria (mainly coryneforms), 13%; filamentous Gram-negative aerobic rods (morphologically akin to Bacillus species), 12%; and yeasts, 7%. Streptomyces was identified among the dominant microflora of only one soil. Nitrogen-fixing bacteria were never isolated, although their presence was indicated in 40% of the soils tested by acetylene-reduction assay. Anaerobes were not detected in any soil using dilutionplate techniques, but were occasionally detected by enrichment in semi-solid media. All of 242 isolates obtained at 10° incubation were able to grow at 15°, 3.3% failed to grow at 18° and a further 9.5% failed to grow at 25°. Of 304 isolates obtained on one-tenth strength Trypticase-Soy agar (0.05% nutrient concentration), 10% failed to grow on the full-strength medium. Soils from the vicinity of Mawson or Davis bases contained a variety of fungi (Cladosporium, Chrysosporium, Acremonium, Phoma, Aureobasidium and Trichoderma species being isolated) in contrast to pristine soils, from which only Verticillium was occasionally isolated.Abbreviations E.C. electrical conductivity - TSA Trypticase-Soy agar - m.S. milli Siemens     

Production of propionic acid by mixed cultures ofPropionibacterium shermanii andLactobacillus casei in autoclave-sterilized whey

Summary Pure cultures ofPropionibacterium freudenreichii ss.shermanii did not grow in autoclave-sterilized cheese whey (121°C, 15 psi, 20 min) at whey concentrations greater than 2% (w/v) spray-dried sweet dairy whey. Propionic acid was produced from autoclave-sterilized whey by growingP. shermanii in mixed culture withLactobacillus casei. In medium containing 5–12% autoclaved whey solids and 1% yeast extract, the mixed culture produced 1.3–3.0% propionic acid, 0.5–1.0% acetic acid, and 0.05–0.80% lactic acid. All the lactose was consumed. Using pH-controlled fermentors (pH=7.0), mixed cultures produced at least 30% more propionic acid than cultures in which pH was not controlled.     

Studies on the distribution of alkalophilic and alkali-tolerant soil fungi II: Fungal flora in two limestone caves in Japan

In a series of studies on the distribution of alkalophilic and alkali-tolerant fungi, soil fungi were isolated from five alkaline calcareous soil samples in two closely located limestone caves (stalactite caves) in Japan using slightly acidic and alkaline media. Some common soil fungi that can grow in alkaline conditions were obtained in high frequencies. The growth response to pH of the isolates revealed that approximately one third (30.8%) of the isolates had the optimum pH in the alkaline range. All isolates of fourAcremonium species and twoChrysosporium species grew well in alkaline conditions, of whichAcremonium sp. andChrysosporium sp. were pronounced alkalophiles. These fungi were thought to be indigenous species in this alkaline environment. The fungal flora in the Japanese alkaline soils was considerably different from the flora reported in alkaline environments in other countries.     

Ergosterol production from molasses by genetically modified <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Ergosterol is an economically important metabolite produced by fungi. Recombinant Saccharomyces cerevisiae YEH56(pHXA42) with increased capacity of ergosterol formation was constructed by combined overexpression of sterol C-24(28) reductase and sterol acyltransferase in the yeast strain YEH56. The production of ergosterol by this recombinant strain using cane molasses (CM) as an inexpensive carbon source was investigated. An ergosterol content of 52.6 mg/g was obtained with 6.1 g/l of biomass from CM medium containing 60 g/l of total sugar in 30 h in shake flask. The ergosterol yield was enhanced through the increasing cell biomass by supplementation of urea to a concentration of 6 g/l in molasses medium. Fermentation was performed in 5-l bioreactor using the optimized molasses medium. In batch fermentation, the effect of agitation velocity on ergosterol production was examined. The highest ergosterol yield was obtained at 400 rpm that increased 60.4 mg/l in comparison with the shake flask culture. In fed-batch fermentation, yeast cells were cultivated, firstly, in the starting medium containing molasses with 20 g/l of total sugar, 1.68 g/l of phosphate acid, and 6 g/l of urea (pH 5.4) for 5 h, then molasses containing 350 g/l of total sugar was fed exponentially into the bioreactor to keep the ethanol level in the broth below 0.5%. After 40 h of cultivation, the ergosterol yield reached 1,707 mg/l, which was 3.1-fold of that in the batch fermentation.     

Esterification of cholesterol and cholestanol in the whole body,tissues, and frass of Heliothis zea

The quantity of free and esterified sterols in the whole body, intestine, hemolymph, fat body, and frass of 6th-instar larvae of H. zea, fed cholesterol or cholestanol, was measured in order to determine if there was a difference in the utilization of these two molecules. The principal sterol in the tissues of the larvae was cholestanol or cholesterol, when they were fed diet containing these two molecules, respectively; there was little, if any, metabolism of dietary cholestanol to cholesterol. There was little or no difference in the amount of total sterol in the whole body, tissues, or frass of larvae fed the two different diets, indicating that the absence of a Δ⁵-bond in cholestanol does not prevent the uptake or distribution of this sterol to various tissues. However, the relative percentage of steryl ester was significantly higher in prepupae reared on a diet containing cholestanol instead of cholesterol (6–7-, 4-, 13-, 4-, and 2-fold increase, for the whole body, intestine, hemolymph, fat body, and frass, respectively). The average percentage of total sterol that was esterified in the tissues was greater in the fat body (10.8 ± 15.4 and 44.2 ± 12.3%, respectively, for larvae fed cholesterol and cholestanol) than in the hemolymph (0.5 ± 0.1 and 6.3 ± 0.8%) and intestine (1.2 ± 0.1 and 4.7 ± 1.1%). The percentage of sterol that was esterified in the frass of larvae was large (26.9 ± 3.7 and 48.2 ± 0.5%, respectively, for larvae fed cholesterol and cholestanol). Therefore, the fact that larvae of H. zea fed cholestanol, instead of cholesterol, contain this saturated molecule as their principal tissue sterol and preferentially esterify it may explain, at least in part, why their rate of growth on cholestanol is slower than on cholesterol.     

Ergosterol from <Emphasis Type="Italic">Phytophthora drechsleri</Emphasis>,a unusual metabolite of a member of this genus

Ergosterol was isolated from the plant pathogenic pseudofungus Phytophthora drechslerigrown on clarified V8 booth (CV8-B). Its structure was confirmed by comparison to an authentic sample. The species was identified by morphological analysis and molecular characterization by PCR: ITS (Internal transcribed spaces). This is the first report of this sterol in Phytophthora.This result is unusual becausePhytophthora fungi were previously thought to be unable to synthesize sterols and the Oomycetes in general do not produce ergosterol.