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A new staining solution for the morphological studies of fungi and Prototheca.

A new staining solution named "PHOL" is described for studying the morphology of clinical and environmental isolates of fungi and Prototheca. In total 69 isolates were used for assessing the efficacy of this newly formulated stain. The solution has shown good ability to stain all 62 isolates of fungi and 7 of Prototheca, and thus helped in the identification of the organisms. The new solution has the potential to stain the young as well as old isolates of fungi and Prototheca. It is emphasized that a wider application of this newly introduced inexpensive and safe staining solution will help the mycologists to study the morphological characteristics of yeast, molds, and Prototheca obtained from man, animals and environment.

Fungi↗

Growth of lignocellulosic-fermenting fungi on different substrates under low oxygenation conditions.

Four soil fungi able to grow under low oxygenation conditions were selected and used in studies to determine the production of enzymes that promote the degradation of lignocellulosic materials. The capacity of these fungi to ferment such materials was also investigated. The fungi were grown in sugarcane bagasse and sawdust at final concentrations of 4 and 10%, as the carbon sources. The strains were cultivated under microaerophilic and combined conditions of oxygenation (aerobic followed by microaerophilic conditions). The results obtained with the basidiomycete specie, Trichocladium canadense, Geotrichum sp., and Fusarium sp. suggest that they prefer lower oxygen concentration for growth and enzyme production. Lignocellulolytic activities were detected in all strains but varied with the carbon source used. The highest levels of these activities were produced by the Basidiomycete specie and Fusarium sp. Ethanol and other nongaseous fermentation products were detected following high-performance liquid chromatography analysis using a supelcogel C-610H column, demonstrating the fermentative capability of these strains. In view of their ability to produce enzymes necessary for the breakdown of lignocellulosic materials and to utilize most of the degradation products for growth, these strains have a great potential for biotechnological application.

Biomass↗

Activity in vitro and in vivo against plant pathogenic fungi of grifolin isolated from the basidiomycete Albatrellus dispansus.

In the course of screening for novel naturally occurring fungicides from mushrooms in Yunnan province, China, the ethanol extract of the fruiting bodies of Albatrellus dispansus was found to show antifungal activity against plant pathogenic fungi. The active compound was isolated from the fruiting bodies of A. dispansus by bioassay-guided fractionation of the extract and identified as grifolin by IR, 1H and 13C NMR and mass spectral analysis. Its antifungal activities were evaluated in vitro against 9 plant pathogenic fungi and in vivo against the plant disease of Erysiphe graminis. In vitro, Sclerotinina sclerotiorum and Fusarium graminearum were the most sensitive fungi to grifolin, and their mycelial growth inhibition were 86.4 and 80.9% at 304.9 microM, respectively. Spore germination of F. graminearum, Gloeosporium fructigenum and Pyricularia oryzae was almost completely inhibited by 38.1microM grifolin. In vivo, the curative effect of grifolin against E. graminis was 65.5% at 304.9 microM after 8 days.

Antifungal Agents↗

Effect of herbizid and touchdown herbicides on soil fungi and on production of some extracellular enzymes.

Glucophilic and cellulose-decomposing fungi were significantly reduced in soil samples treated with 0.019-0.152 mg a.i./kg soil of the herbicides Herbizid and Touchdown. The decrease was regularly correlated with the doses of the two herbicides and persisted till the end of the experiment (12 weeks). The isolated fungi were found to be able to produce hydrolytic extracellular enzymes in solid media but with variable capabilities. The ability to produce enzymes was adversily affected by the incorporation of herbicides in culture media. Lower doses of herbicides were occasionally promotive to enzyme production and mycelial growth of some fungi. Incorporation of 50 ppm of Herbizid and Touchdown significantly activated amylase production and mycelial dry weight in cultures of Fusarium oxysporum, Mucor hiemalis and Penicillium chrysogenum. There was a significant increase in C1-cellulase produced by F. oxysporum and P. aurantiogriseum when cultures were treated with 50, 100 and 200 ppm of Herbizid which induced also more Cx-cellulase production by P. chrysogenum. Lipase and protease production was always lower in treated than in control fungal cultures.

Amylases↗

Therapy of infections caused by dematiaceous fungi.

Dematiaceous fungi are responsible for a wide variety of clinical syndromes, from local infections due to trauma, to disseminated infection in immunocompromised patients. These fungi are unique owing to the presence of melanin in their cell walls, which imparts the characteristic dark color to their spores and hyphae. Melanin may also be a virulence factor in these fungi. Therapy depends upon the clinical syndrome. Local infection may be cured with excision alone, while systemic disease is often refractory to therapy. Azoles such as itraconazole and voriconazole have the most consistent in vitro activity, although newer agents may also play a role in therapy in the future.

Antifungal Agents↗

Virulence factors in fungi of systemic mycoses.

Pathogenic fungi that cause systemic mycoses retain several factors which allow their growth in adverse conditions provided by the host, leading to the establishment of the parasitic relationship and contributing to disease development. These factors are known as virulence factors which favor the infection process and the pathogenesis of the mycoses. The present study evaluates the virulence factors of pathogenic fungi such as Blastomyces dermatitidis, Coccidioides immitis, Cryptococcus neoformans, Histoplasma capsulatum and Paracoccidioides brasiliensis in terms of thermotolerance, dimorphism, capsule or cell wall components as well as enzyme production. Virulence factors favor fungal adhesion, colonization, dissemination and the ability to survive in hostile environments and elude the immune response mechanisms of the host. Both the virulence factors presented by different fungi and the defense mechanisms provided by the host require action and interaction of complex processes whose knowledge allows a better understanding of the pathogenesis of systemic mycoses.

Cell Adhesion Molecules↗

[Airborne fungi and sensitization in atopic individuals in Porto Alegre, RS, Brazil].

OBJECTIVE: To identify and quantify the fungal spores in the atmospheric air of Porto Alegre and to assess sensitization to these allergens in atopic individuals suffering from rhinitis and asthma. METHODS: A total of 52 samples were obtained using a Rotorod Sampler from April 2000 through March 20001. Allergy skin tests with anemophilous fungus allergens as well as fluorimmunoenzymatic tests were performed in 39 atopic individuals with rhinitis and asthma in order to detect the incidence of respiratory allergies caused by these airborne fungi. RESULTS: In the 3773 fungal spores detected in this study, there was a prevalence of ascosporos (50.49%), Cladosporium (17.86%), Aspergillus/Penicillium (15.03%), basidiosporos (3.84%), rusts (3.82%), and Helminthosporium (2.49%), as well as a lesser frequency of Botrytis (1.22%), Alternaria (1.19%), smuts (0.90%), Curvularia (0.87%), Nigrospora (0.61%), and Fusarium (0.08%). Sixty spores (1.59%) detected here could not be identified by the systematic key used. Among the atopic individuals tested, 15.38% were found to be sensitized by these anemophilous fungi allergens. CONCLUSIONS: The occurrence of a great number of fungal spores emphasizes the importance of studying anemophilous fungi locally, since they are responsible for 15.38% of allergic sensitization in atopic individuals with asthma and rhinitis.

Air Microbiology↗

[Pathogenicity of hyphomycet fungi to aphids Aphis gossypii Glover and Myzus persicae (Sulzer) (Hemiptera: Aphididae)].

The aphids Aphis gossypii and Myzus persicae are cosmopolitan, poliphagous and damage cultivated plants. The effects of the entomopathogenic fungi Beauveria bassiana (isolate IBCB 66), Metarhizium anisopliae (isolate IBCB 121), Paecilomyces fumosoroseus (isolate IBCB 141) and Lecanicillium (= Verticillium) lecanii (isolate JAB 02) on third instar nymphs of A. gossypii and M. persicae were evaluated in the laboratory at 25 degrees C, 70 +/- 10% RH and 12h photophase. The aphids were transferred to petri dishes with a foliar disk (cotton or pepper) with a layer of 1 cm tick of agar-water. The fungi were applied in a suspension containing 1.0 x 106 to 1.0 x 108 conidia/ml. In the control treatment 1 ml of sterilized water was added to the foliar disks. The mortality of aphids was evaluated daily. B. bassiana and M. anisopliae caused 100% mortality at the seventh day after inoculation, for both species. L. lecanii was the fungus that provided mortality later in the aphids and M. persicae was more susceptible to both fungi than A. gossypii.

Animals↗

Pathogenicity of entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae to Ixodidae tick species Dermacentor variabilis, Rhipicephalus sanguineus, and Ixodes scapularis.

Nymphal and adult ticks from three different tick species, Dermacentor variabilis Say, Ixodes scapularis Say, and Rhipicephalus sanguineus Latrielle, were treated with conidia and blastospores of the entomopathogenic fungi Beauveria bassiana (Bals.) Vuill. and Metarhizium anisopliae Metschnikoff. Dose-response experiments indicated that a critical concentration of fungal spores is required for infection and mortality. Over a 28-d time course, fungal suspensions of either B. bassiana or M. anisopliae at 10(8) conidia/ml resulted in 50-70% mortality in adult I. scapularis and R. sanguineus, but <20% mortality in D. variabilis ticks. R. sanguineus nymphs were highly susceptible to both entomopathogenic fungi, displaying >60% mortality within 14 d postinfection and >90% mortality within 21-28 d postinfection. D. variabilis nymphs also were more susceptible than their corresponding adults, displaying mortalities ranging from 20 to 40% 28 d postinfection. I. scapularis nymphs, however, seemed to be slightly less susceptible than adults (45% mortality, 28 d postinfection). The addition of nutrients to fungal cell suspensions did not have any noticeable effects on mortality toward any of the tick species tested. Significant mortality against D. variabilis adults (approximately 65%) was noted only when B. bassiana fungal cells with growth media carryover were used as the inoculum against the ticks. Entomopathogenic fungi such as B. bassiana and M. anisopliae may have the potential for controlling populations of I. scapularis and R. sanguineus, and under certain conditions D. variabilis. Our results indicate that inoculum conditions can greatly affect successful virulence and subsequent mortality.

Animals↗

Recombinant DNA in filamentous fungi: progress and prospects.

Recombinant DNA technology enables the creation of well-defined alterations in the genetic material of an organism. Methods to manipulate recombinant DNA in the filamentous fungi (a group of microorganisms that includes species of academic as well as commercial interest) have recently been developed. This has been the result of adaptation of procedures successfully employed in the manipulation of other microorganisms. There are a number of similarities in the behavior of recombinant DNA in different fungi, but a number of differences have also been observed between the filamentous and the nonfilamentous fungi. Such differences include the ability to identify DNA replication origins and the host range of expression of fungal genes.

Cloning, Molecular↗

Exposure levels of airborne bacteria and fungi in Korean swine and poultry sheds.

There is a limited amount of information available on stock farmers' exposure to airborne bacteria and fungi. In this study, the authors measured the airborne bacteria and fungi concentrations 46 times inside two swine sheds and four poultry sheds. Aspergillus, Cladosporium, and Penicillium were the most prevalent fungal genera, as most of the fungi belonged to these groups. Many microbial concentrations exceeded the Korean indoor bioaerosol guideline 800 CFU m(-3) (i.e., colony-forming units per cubic meter of air). This finding suggests the need for a strategy to reduce stock farmers' occupational exposure to the microorganisms in swine and poultry sheds, and the necessity of performing a longitudinal survey to better examine farmer exposure levels and their variability. Another finding was that the use of a sanitary mask may reduce stock farmers' exposure to bioaerosols.

Agricultural Workers' Diseases↗

Colonization and succession of fungi during decomposition of Swida controversa leaf litter.

Decomposition processes of Swida controversa leaves were investigated in initially sterilized (fungi-excluded) and nonsterilized freshly fallen leaves to examine the relationship between chemical changes and fungal succession during decomposition and the effect of exclusion of previously established phyllosphere fungi from leaves on subsequent decomposition and fungal succession. Fifteen species were isolated frequently from decomposing leaves with surface-disinfection and washing methods. These fungi were divided into early and late colonizers according to their occurrence during decomposition. The 1.5 y decomposition process was divided into three stages characterized by different dominant organic chemical constituents. A clear relationship was demonstrated between chemical changes and fungal succession. Total hyphal length and frequencies of some early colonizers were reduced in initially sterilized leaves at 3 wk, but this had no significant effect on loss of litter mass or chemical changes during the first 3 wk or on the subsequent decomposition and fungal succession.

Biomass↗

Possible involvement of pleiomorphic vacuolar networks in nutrient recycling in filamentous fungi.

Morphological analyses of vacuoles in filamentous fungi in the past decade have led to the remarkable finding that they are highly pleiomorphic organelles. Among them, tubular vacuoles have been implicated in nutrient transport between hyphal tips and the host plant surface in mycorrhizal fungi. However, a series of works suggested the presence of tubular vacuoles in other fungi that are not mycorrhizal, including Aspergillus oryzae, hinting at more general roles of the tubular vacuoles. Recently, we made two key observations by using the fusion protein of enhanced green fluorescent protein (EGFP) with a putative vacuolar t-SNARE in A. oryzae; tubular vacuoles formed more extensively in hyphae that were not in contact with nutrients, and vacuoles that were interconnected by tubules in the mature mycelial region displayed traces of microautophagy-mediated degradation of cytoplasm. The aim of this addendum is to discuss the possible involvement of vacuoles in degrading, transporting, and recycling nutrients from the mature mycelial region to hyphal tips, to support the continuous tip growth.

Aspergillus oryzae↗

Fungi, aflatoxins, and cyclopiazonic acid associated with peanut retailing in Botswana.

Peanuts are important food commodities, but they are susceptible to fungal infestation and mycotoxin contamination. Raw peanuts were purchased from retail outlets in Botswana and examined for fungi and mycotoxin (aflatoxins and cyclopiazonic acid) contamination. Zygomycetes were the most common fungi isolated; they accounted for 41% of all the isolates and were found on 98% of the peanut samples. Among the Zygomycetes, Absidia corymbifera and Rhizopus stolonifer were the most common. Aspergillus spp. accounted for 35% of all the isolates, with Aspergillus niger being the most prevalent (20.4%). Aspergillus flavus/parasiticus were also present and accounted for 8.5% of all the isolates, with A. flavus accounting for the majority of the A. flavus/parasiticus identified. Of the 32 isolates of A. flavus screened for mycotoxin production, 11 did not produce detectable aflatoxins, 8 produced only aflatoxins B1 and B2, and 13 produced all four aflatoxins (B1, B2, G1, and G2) in varying amounts. Only 6 of the A. flavus isolates produced cyclopiazonic acid at concentrations ranging from 1 to 55 microg/kg. The one A. parasiticus isolate screened also produced all the four aflatoxins (1,200 microg/kg) but did not produce cyclopiazonic acid. When the raw peanut samples (n = 120) were analyzed for total aflatoxins, 78% contained aflatoxins at concentrations ranging from 12 to 329 microg/kg. Many of the samples (49%) contained total aflatoxins at concentrations above the 20 microg/kg limit set by the World Health Organization. Only 21% (n = 83) of the samples contained cyclopiazonic acid with concentrations ranging from 1 to 10 microg/kg. The results show that mycotoxins and toxigenic fungi are common contaminants of peanuts sold at retail in Botswana.

Aflatoxins↗

Sampling of high concentrations of airborne fungi.

The paper discusses a method developed for determining airborne fungi particles in environments highly contaminated with mold fungi. The collection of airborne fungi was performed with two slit samplers. These sampling devices were found to give the highest values out of the three different types tested simultaneously. After spores had been collected with the slit samplers, the collection medium, an agar gel, was removed from the petri dishes and homogenized in a sterile 0.9% sodium chloride solution. The homogenate was diluted stepwise and spread on agar plates prior to the cultivation and the determination of viable counts. When the homogenization procedure was tested on samples collected from three different work environments, no increase or decrease in the number of colony-forming units could be detected. Storage of the homogenate at 2 degrees C over 9 d did not increase the number of viable fungal colonies.

Air Microbiology↗

Efficacy of disinfectants against fungi isolated from skin and nail infections.

Commercial disinfectants classified as fungicides may not be effective against commonly encountered fungi within reasonable periods. Cell suspensions of clinical fungal isolates were exposed to use-dilutions of various disinfectants. Quaternary ammonium compounds, iodophors, and phenolics were not fungicidal against all test fungi within 60 min of exposure. Trichophyton mentagrophytes, Epidermophyton floccosum, and Aspergillus fumigatus were among the more resistant fungi. Disinfectants that possess low-level activity should not be used for disinfection of medical instruments that come in contact with the patient. The only reliable and safe measure is to use high-level disinfectants such as the glutaraldehydes, which are fungicidal in 15 to 30 min.

Disinfectants↗

A three-year study of viable airborne fungi in the North Carolina Zoological Park R.J.R. Nabisco Rocky Coast Alcid Exhibit.

Aspergillosis is a common cause of mortality in captive birds, particularly in recently imported birds or captive chicks and their parents. Use of the Andersen N-6 single-stage viable air sampler in the North Carolina Zoological Park (NCZP) R.J.R. Nabisco Rocky Coast Alcid Exhibit before and after the introduction of birds allowed a unique study of the mycological content of the air in a developing self-contained ecosystem. The Alcid Exhibit had a median count of 17 colony-forming-units (CFU)/m3 of air in comparison to 200-500 CFU/m3 and 1,000-3,500 CFU/m3 reported in human dwellings and the NCZP R.J. Reynolds Forest Aviary, respectively. Cladosporium and Penicillium represented 21.3% and Aspergillus 1.08% of the fungi collected. During the study, no respiratory mycoses were reported in any of the alcids. Continuous high-efficiency particulate air filtration, maintenance of low exhibit air temperatures, and an environment with little residual organic material capable of supporting fungal growth were important factors contributing to low colony counts. All colony counts >100 CFU/m3 in the exhibit were related to the apparent introduction of fungi from outside the facility. A reduction in the number of fungi transported from an external source into enclosed cool-temperature aviaries may be sufficient to avoid outbreaks of aspergillosis.

Air Microbiology↗