Comparison of methods for isolation of dematiaceous fungi from nature

Direct plating and animal inoculation techniques were compared for effectiveness in isolating dematiaceous fungi from nature. The direct plating technique involved comparison of aqueous and mineral oil extraction of the samples with subsequent plating on Mycobiotic and Sabhi agar. Twenty four different organisms were recovered from 19 samples using both extraction procedures and both media. Dematiaceous fungi isolated by direct plating were Alternaria alternata, Cladosporium spp., Exophiala jeanselmei, Wangiella dermatitidis and 6 unidentified organisms. Direct plating resulted in more isolations of dematiaceous fungi partly because of the frequent isolations of A. alternata and W. dermatitidis. Both more and a larger variety of organisms were recovered on Mycobiotic than on Sabhi agar. The aqueous extraction of samples resulted in more direct plating isolations of fungi than oil extraction.Only dematiaceous fungi were isolated with the animal inoculation techniques. Fungi isolated from hamsters included Cladosporium spp., Exophiala spinifera, Phialophora verrucosa, Rhinocladiella sp. and 3 unidentified organisms. Fungi isolated by the mouse inoculation technique included Bispora betulina, Cladosporium spp., W. dermatitidis and 1 unidentified organism. In general there was variation in the types of organisms isolated from the same samples depending on the isolation procedure used.     

Coccidioides immitis isolated from armadillos (Dasypus novemcinctus) in the state of Piauí, northeast Brazil

Natural infection of armadillos with Coccidioides immitis was studied in the state of Piauí, northeast of Brazil, endemic for coccidioidomycosis. In 1998, 26 nine-banded armadillos (Dasypus novemcinctus) were captured in 4 different counties. The animals were sacrificed under deep anesthesia with ether. At necropsy fragments of spleen, liver, lungs and heart were homogenized and seeded onto Sabouraud dextrose agar with and without cycloheximide (BBL, USA). Part of each organ was also processed for histological examination. Suspected colonies of filamentous fungi observed after the second week of incubation at room temperature, exhibiting barrel-shaped arthroconidia alternating with empty spaces, were inoculated intraperitoneally into mice. Three armadillos proved to be infected with C. immitis. Mice inoculated with suspected colonies obtained from homogenized spleen of three and liver of two armadillos developed disseminated coccidioidomycosis and immature and mature spherules of C. immitis were disclosed in several organs. For the first time armadillos (D. novemcinctus) were found naturally infected with C. immitis, adding new data on the ecology and on a possible role of these ancestral mammals in the evolutionary life cycle of this fungus. This revised version was published online in July 2006 with corrections to the Cover Date.     

Fungal flora and mycotoxins of six kinds of nut seeds for human consumption in Saudi Arabia

A wide range of moulds representing several genera and species, was recorded in this study from 5 seed samples of each almond, cashew nut, chestnut, hazelnut, pistachio nut and walnut collected from different markets in Ar' Ar, Saudi Arabia. The total counts of fungi were widely fluctuated between 1960–7704 and 1948–7434 colonies/g dry seeds on glucose-Czapek's and glycerol agar media at 28°C, respectively, and represented twenty genera, 53 species and 2 varieties of fungi. The prevalent fungi on the 2 agar media wereAspergillus flavus, A. niger andPenicillium chrysogenum. On glucose-Czapek's agar,Rhizopus stolonifer andAspergillus flavus var.columnaris were isolated from all 6 kinds of nut,A. parasiticus from 5 kinds andA. fumigatus from 4 kinds with high frequencies.Eurotium species were completely absent on glucose-Czapek's agar but they were isolated in high frequency from all kinds of nut on glycerol agar medium. The different nut samples were analyzed by thin layer chromatography for the presence of aflatoxins B₁, B₂, G₁ & G₂, citrinin, ochratoxins, patulin, sterigmatocystin, diacetoxyscirpenol, T-2 toxin and zearalenone. Aflatoxins B₁ & G₁ were detected in 3 out of the 5 samples tested of chestnut at concentrations ranging between 20 to 60 µg/kg. All other samples of almond, cashew nut, hazelnut, pistachio nut, and walnut that were analyzed were mycotoxin free.     

Efficacy of brain heart infusion-egg albumen agar,yeast extract phosphate agar and peptone glucose agar media for isolation of Blastomyces dermatitidis from sputum

The efficacy of brain heart infusion (BHI)-egg albumen agar, yeast extract phosphate agar and several modified peptone glucose agar media was evaluated for isolation of Blastomyces dermatitidis from sputum concomitantly seeded with the yeast form of the pathogen and Candida albicans. Based upon high per cent culture positivity of sputum, improved recovery (CFU/ml) of the seeded inoculum, faster growth rate of B. dermatitidis and low level of contamination, BHI-egg albumen agar, followed by yeast extract phosphate agar are recommended as the media of choice for the isolation of B. dermatitidis from contaminated clinical specimens.     

Selective Degradation of Wood Components by White-Rot Fungi

In order to find naturally occurring white-rot fungi which preferentially degrade lignin. 25 different species of such fungi were cultivated on pine wood blocks and on kraft lignin agar plates with and without cellulose. Due to differences in phenol oxidase reactions on the kraft lignin agar plates, the 25 fungi could be divided into two groups, 1 and 2, which also differed in other properties. The three Group I fungi Sporotrichum pulverulentum, Phanerochaete sp. L1 and Polyporus dichrous produced high levels of endo-l,4-β-glucanase and cellobiose:quinone oxidoreductase in shaking cellulose flasks and a low level of phenol oxidase in standing wood meal flasks, The four fungi Merulius tremellosus, Phlebia radiata, Pycuoporus cinnabarinus and Pleurotus ostreatus from Group 2, on the other hand, produced low levels of endo-1,4-β-glucanase and cellobiose:.quinone oxidoreductase in the cellulose. flasks and a high level of phenol oxidase in the wood meal flasks. Analyses of pine wood blocks degraded by the above-mentioned fungi in the presence of either malt extract, asparagine or NH₄H₂PO₄ revealed that malt extract gave good lignin degradation. In the presence of this nutrient source. P. cinnabarinus, at 3.4% weight loss, even degraded 12.5% lignin without loss of cellulose or mannan. No common degradation pattern was, however, obtained using mall extract, asparagine or NH₄H₂PO₄, It is suggested that while-rot fungi, which preferentially degrade lignin, may be found among Group 2 fungi producing large amounts of phenol oxidases.     

Effects of an organophosphorus insecticide on the growth and cellulolytic activity of fungi

The organophosphorus insecticide Selecron [O-(4-bromo-2-chlorophenyl) O-ethyl S-n-propyl-phosphorotioate] at 10 and 50 ppm significantly decreased respiration, mycelial protein, extracellular protein and mycelial dry weight of Aspergillus fumigatus, A. terreus and Myceliophthora thermophila when grown at 45°C. C_x and C₁ cellulases of tested fungi were significantly decreased. However, C₁ cellulase of A. fumigatus was slightly increased.     

Effects of fungicides on Aspergillus fumigatus

Aspergillus fumigatus was the most frequently isolated thermophilous fungus from green leaf surfaces. The application of fungicides significantly reduced the frequency of its occurrence there. A. fumigatus was relatively tolerant to fungicides. On Captan-, Thiram-, and Verdasan-treated leaves, A. fumigatus constituted 66%–80% of the total number of isolates obtained at 45°C from each treatment while Dicloran did not depress the percentages. At 45°C, A. fumigatus was found to be strongly cellulolytic with a slow rate of radial extension on YpSs agar and rapid rate of mycelial growth in Czapek Dox liquid medium. Increasing concentrations of all four fungicides reduced or prevented growth, sporulation, starch depletion and cellulose clearing of A. fumigatus. The fungus could tolerate higher concentrations of HgCl₂ than of Verdasan. 0.5 g/ml of the four fungicides altered the rates of mycelial growth but not the maximum amount of mycelial dry weight attained.     

Hydroxylamine oxidation and subsequent nitrous oxide production by the heterotrophic ammonia oxidizer Alcaligenes faecalis

Nitrous oxide (N₂O), a greenhouse gas, is emitted during autotrophic and heterotrophic ammonia oxidation. This emission may result from either coupling to aerobic denitrification, or it may be formed in the oxidation of hydroxylamine (NH₂OH) to nitrite (NO₂ ⁻). Therefore, the N₂O production during NH₂OH oxidation was studied with Alcaligenes faecalis strain TUD. Continuous cultures of A. faecalis showed increased N₂O production when supplemented with increasing NH₂OH concentrations. ¹⁵N-labeling experiments showed that this N₂O production was not due to aerobic denitrification of NO₂ ⁻. Addition of ¹⁵N-labeled NH₂OH indicated that N₂O was a direct by-product of NH₂OH oxidation, which was subsequently reduced to N₂. These observations are sustained by the fact that NO₂ ⁻ production was low (0.23 mM maximum) and did not increase significantly with increasing NH₂OH concentration in the feed. The NH₂OH-oxidizing capacity increased with increasing NH₂OH concentrations. The apparent V _max and K _m were 31 nmol min⁻¹ mg dry weight⁻¹ and 1.5 mM respectively. The culture did not increase its growth yield and was not able to use NH₂OH as the sole N source. A non-haem hydroxylamine oxidoreductase was partially purified from A. faecalis strain TUD. The enzyme could only use K₃Fe(CN)₆ as an electron acceptor and reacted with antibodies raised against the hydroxylamine oxidoreductase of Thiosphaera pantotropha. Received: 1 September 1998 / Received revision: 5 November 1998 / Accepted: 7 November 1998     

Analysis of Secondary Metabolites of Microscopic Fungi of the Genus Penicillium by Chromatographic Techniques

Combinations of various systems of thin-layer chromatography and high-performance liquid chromatography (HPLC) were efficient in analyzing 39 nitrogen-containing secondary metabolites (alkaloids) produced by 12 strains of microscopic fungi of the genus Penicillium. Chromatographic mobility of alkaloids on Silufol plates was determined in the following systems: (a) chloroform, methanol, and 25% NH₄OH (90 : 10 : 1, 90 : 10 : 0.1, or 80 : 20 : 0.2); (b) chloroform and acetone (9 : 1); and (c) ethyl acetate, methanol, and 25% NH₄OH (85 : 15 : 10); staining was performed using Ehrlich's reagent. Conditions for separation of clavine alkaloids by HPLC on Spherisorb ODS-2 and Supelcosil LC-18 columns (gradient elution) were optimized. Retention values of 22 alkaloids were compared to those of agroclavine and roquefortine.     

Recovery of endophytic Beauveria bassiana on a culture medium based on cetyltrimethylammonium bromide

Selective media (potato dextrose agar plus yeast extract, different concentrations of cetyltrimethylammonium bromide (CTAB) and different antibacterials) were evaluated for the isolation of endophytic Beauveria cf. bassiana from dry bean (Phaseolus vulgaris) plants. CTAB amounts of 0.15 and 0.3?g/L plus either dihydrostreptomycin, oxytetracycline or doxycycline resulted in the lowest numbers of common saprophytic fungi and the highest proportional recovery of Beauveria colonies from internal plant tissues.     

The genus Blastomycoides as a mycological entity

Summary Studies were made on some environmental factors promoting yeast-phase growth ofBlastomycoides in chemically defined media. With inclusion of different tryptophane desaminase inhibitors, pure yeast phase of growth was obtained. The same factors facilitate the development of perfect sexual forms and the observation of the cytological characteristics. Therefore,Blastomycoides, represented byBlastomyces dermatitidis, Bl. brasiliensis, Bl. cerolytica andBl. immitis (formerlyCoccidioides immitis), form a special genus which is very near to the Protomyces and represents a transitory phase between the Endomycetales and the Taphrinales. Although the vegetative mycelium of the Blastomycoides resembles the Aleurismaceae, they cannot be classified as such, especially because they are dimorphic fungi (filamentous-yeast) and their sexual forms are characteristic asci on a very primitive level.Blastomycoides not only represent a group of fungi causing grave visceral mycoses (blastomycoses) but they also are an indiscutable mycological entity, as yet represented in the genusBlastomycoides     

Decolorization of a dye industry effluent by <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> XC6

The strain Aspergillus fumigatus XC6 isolated from mildewing rice straw was evaluated for its ability to decolorize a dye industry effluent. The strain was capable of decolorizing dyes effluent over a pH range 3.0–8.0 with the dyes as sole carbon and nitrogen sources. The optimum pH was 3.0; however, supplemented with either appropriate nitrogen sources (0.2% NH₄Cl or (NH₄)₂SO₄ ) or carbon sources (1.0% sucrose or potato starch), the strain decolorized the effluent completely at the original pH of the dyes effluent. Therefore, A. fumigatus XC6 is an efficient strain for the decolorization of reactive textile dyes effluents, and it might be a practical alternative in dyeing wastewater treatment.     

Dot-immunobinding assay in the detection of IgG antibodies against farmer's lung antigens

Circulating antibodies against Faenia rectivirgula, Thermoactinomyces candidus, T. vulgaris and Aspergillus fumigatus were studied in the sera of 14 clinically proven farmer's lung patients and 10 normal controls using three immunological methods. These methods were agar gel double diffusion (DD), biotin-avidin-linked immunosorbent assay (BALISA) and dot-immunobinding assay (DIBA). Agar gel diffusion, the least sensitive of the three methods, failed to detect antibodies in some of the patients, while BALISA detected antibodies even in the normal controls. However, the sensitivity of dot-immunobinding assay was in between DD and BALISA while the specificity was comparable to DD to all the antibodies except against A. fumigatus antigens. Dot-immunobinding assay gave faster results than DD and the blots can be stored as record for longer periods of time without fading.     

Artificial diet for continuous maintenance of the maritima earwig Anisolabis maritima (Bonelli) (Dermaptera: Carcinophoridae)

Anisolabis maritima is an important predator for the eggs of the red palm weevil Rhynchophorous ferrugineus. It could be successfully reared in the laboratory, on an artificial diet composed of dry kidney beans, Brewers yeast, chicken, egg yolk, agar, ascorbic acid, mould inhibitors, vitamin B₁₂, folic acid and riboflavin.     

Effects of tissue storage and freezing on brain glutamate uptake

Glutamate uptake appears to be stable when measured in rat striatal synaptosomes from tissue stored for up to four hours post-mortem at 25°C. Between four and eight hours storage at room temperature there is a sharp 70% decrease in uptake. Freezing of tissue on dry ice, storage at 4°C for up to 7 days and at ?80°C for 5 days results in 20–30% residual glutamate uptake. Quantitatively similar data can be obtained in eight extrastriatal brain areas. Kinetic analysis of glutamate uptake in stored and frozen tissue reveals the loss of the majority of both sodium-dependent high affinity and temperature-sensitive low affinity sites (v_max-values) while the respective K_m-values are not significantly changed. Pharmacological properties of the high affinity uptake versus a number of specific and metabolic uptake inhibitors remain unaltered by the storage and freezing procedure. The tissue treatment chosen for the present study roughly corresponds with the preparation of human post-mortem brain tissue for enzyme-, receptor-binding- or neuro-transmitter assays. It therefore seems conceivable that meaningful uptake studies can be performed on human autopsy material, thus adding an important parameter to the battery of neurochemical markers already accessible for post-mortem $\text{in}$$\text{vitro}$ examination.     

Elastase activity of fungi with anamorphs similar to Coccidioides immitis

The elastin digestion assay was examined to determine if it would facilitate the identification of Coccidioides immitis when non-pathogenic fungi resembling C. immitis are encountered. Fungal isolants tested have anamorphs that closely resemble the macroscopic or microscopic morphology of C. immitis. Elastin hydrolysis was measured by elastin-agar plate assays. Approximately 80% of the isolants hydrolyzed elastin; thus, the elastin digestion assay as a differential test appears to have little value.     

Survey of the mycoflora and mycotoxins of cotton seeds and cotton seed products in Egypt

Thirty-nine species and 16 fungal genera were isolated from Egyptian cotton seeds, cotton seed meal and cotton seed cake on 1% glucose-Czapek's agar medium incubated at 28 °C. Aspergillus was the most frequent genus and it emerged in 87–100% of the samples contributing 70–98% of total fungi in the three substrates tested. The most common species were A. niger, A. flavus, A. fumigatus, A. terreus and Rhizopus stolonifer; A. niger, A. fumigatus and Penicillium corylophilum; and A. niger, A. flavus, A. terreus, A. nidulans and Rhizopus stolonifer, respectively. Cotton seeds and cotton seed products were naturally contaminated by aflatoxin B₁ and B₂. About 16% of the different substrates tested were positive for aflatoxin contamination. No citrinin, ochratoxin A, patulin, sterigmatocystin, diacetoxyscirpenol, T-2 toxin or zearalenone were detected in the samples assayed.     

Studies on an interesting Saccharomyces carlsbergensis mutant

Summary 32 sputa from 29 patients known to have yeasts were emulsified and quantitatively plated on Sabouraud's glucose agar. Colony counts showed that the yeast flora varied from about 300 to about one million cells per ml of the specimen. Sputa collected at bed-side and plated within 2 hours gave lower counts than those received routinely. In the 28 specimens in which identification was carried outC. albicans was found in 23 as the only or the dominant species andC. tropicalis in 4; an unidentified species was present in one specimen.This work was supported by a Grant from the Medical Research Council, Ottawa, Canada.     

Coccidioidin sensitivity in control,immunized and infected guinea pigs

Summary Delayed hypersensitivity to potent coccidioidin developed in guinea pigs immunized with deadC. immitis arthrospores and guinea pigs infected with an aerosol ofC. immitis arthrospores at weeks 1 and 2. Delayed hypersensitivity in control animals sensitized by repeated intradermal testing developed at weeks 3 and 4. The delayed hypersensitivity responses were characterized grossly by indurations larger than 25 mm² and could be seen at 6 and 24 hours after testing. Retesting reduced the size of the 24 hour indurations when compared to virginal reactions. The retest delayed reactions in infected animals had indurations at 24 and 48 hours that were larger than those in the other groups.In those animals that were skin test positive but not challenged no tube precipitins, agar gel precipitins, CF antibodies, anaphylaxis or immediate hypersensitivity were detected. Because of the inability to detect precipitins the early phase of the hypersensitivity seen at 6 hours was not considered an Arthus reaction.     

Ubiquinone system,GC contents and cellular fatty acid composition of species of the form-genusMalbranchea andCoccidioides immitis for chemotaxonomic study

Ubiquinone system, GC contents and cellular fatty acid (CFA) composition were analyzed as criteria for chemotaxonomy ofMalbranchea species andCoccidioides immitis, which are suggested to be phylogenetically related. Based on the major ubiquinone,Malbranchea spp. were divided into two groups, of which one group possessed the same major ubiquinone asC. immitis. Similar GC contents and CFA profiles were obtained for the species ofMalbranchea andC. immitis. On the basis of these criteria the relationships between the fungi are discussed.