Fungal flora associated with combine harvester wheat and sorghum dusts from Egypt

107 species and 8 species varieties belonging to 44 genera were collected from combine harvester wheat and sorghum dusts (35 genera and 91 species + 4 varieties) and from the atmosphere of their hay sites (26 genera and 69 species + 4 varieties) on glucose- and cellulose-Czapek's Dox agar at 28 degrees C and 45 degrees C. The mycoflora of wheat and sorghum dusts were basically similar on the two types of media and the most common fungi were: Alternaria alternata, Aspergillus flavus, A. fumigatus, A. niger, A. ochraceus, A. sydowii, A. terreus, Cochliobolus spicifer, Emericella nidulans, Fusarium moniliforme, Penicillium chrysogenum, P. duclauxii, P. funiculosum and P. oxalicum. Truly thermophilic species were frequently encountered from the two substrates: Chaetomium thermophilum, Humicola grisea var. thermoidae, H. insolens, Malbranchea pulchella var., sulphurea, Rhizomucor pusillus, Sporotrichum thermophilum, Talaromyces thermophilus, Thermoascus thermophilus and Thermomyces lanuginosus. The airborne fungi in the two atmospheres were basically similar and the most prevalent species were members of Alternaria (1 species), Aspergillus (18 species + 2 varieties), Chaetomium (2 species), Cochliobolus(3 species), Emericella (3 species + 2 varieties), Fusarium (3 species), Mucor (1 species), Penicillium (14 species) and Stachybotrys (1 species).     

cDNA cloning, biological and immunological characterization of the alkaline serine protease major allergen from Penicillium chrysogenum

BACKGROUND: Penicillium chrysogenum (Penicillium notatum) is a prevalent airborne Penicillium species. A 34-kD major IgE-reacting component from P. chrysogenum has been identified as an alkaline serine protease (Pen ch 13, also known as Pen n 13 before) by immunoblot and N-terminal amino acid sequence analysis. METHODS: In the present study, Pen ch 13 was further characterized in terms of cDNA cloning, protein purification, enzymatic activity, histamine release and IgE cross-reactivity with alkaline serine protease allergens from two other prevalent fungal species--P. citrinum (Pen c 13) and Aspergillus flavus (Asp fl 13). RESULTS: A 1,478-bp cDNA (Pen ch 13) that encodes a 398-amino-acid alkaline serine protease from P. chrysogenum was isolated. This fungal protease has pre- and pro-enzyme sequences. The previously determined N-terminal amino acid sequence of the P. chrysogenum 34-kD major allergen is identical to that of residues 116-125 of the cDNA. Starting from Ala116, the deduced amino acid sequence (283 residues) of the mature alkaline serine protease has a calculated molecular mass of 28.105 kD with two cysteines and two putative N-glycosylation sites. It has 83 and 49% sequence identity with the alkaline serine proteases from P. citrinum and A. fumigatus, respectively. The recombinant Pen ch 13 was recovered from inclusion bodies and isolated under denaturing condition. This recombinant protein reacted with IgE antibodies in serum from an asthmatic patient and with monoclonal antibodies (PCM8, PCM10, PCM39) that reacted with the 34-kD component from P. chrysogenum. The N-terminal amino acid sequence of the purified native Pen ch 13 is identical to that determined previously for the 34-kD major allergen in crude P. chrysogenum extracts. The purified native Pen ch 13 has proteolytic activity with casein as the substrate at pH 8.0. This enzymatic activity was inhibited by phenylmethylsulfonyl fluoride or diethylpyrocarbonate. Pen ch 13 was also able to degrade gelatin and collagen but not elastin. Basophils from 5 asthmatic patients released histamine (12-73%) when exposed to the purified Pen ch 13. In ELISA (enzyme-linked immunosorbent assay) experiments, IgE for Pen ch 13 was able to compete with purified Pen ch 13, Pen c 13 or Asp fl 13 in a dose-related manner. CONCLUSIONS: These results demonstrated that the 34-kD major allergen of P. chrysogenum is an alkaline serine protease. These results also indicated that atopic patients primarily sensitized by either of these prevalent fungal species may develop allergic symptoms by exposure to other environmental fungi due to cross-reacting IgE antibodies against this protease.     

Studies on the mycoflora of Aswan High Dam Lake, Egypt: monthly variations

Fifty-one species and one variety appertaining to twenty one genera of mesophilic fungi were recovered from the monthly samples of marginal water (44 species, 1 variety and 18 genera) and submerged mud (78 species, 1 variety and 30 genera) of Aswan High Dam Lake during the period from July 1985 to December 1986. The most common species were Aspergillus fumigatus, A. flavus, A. terreus, A. niger and Penicillium funiculosum. The highest fungal populations were almost detected either in October, in December 1985 or in February 1986. Of the 12 thermophilic and thermotolerant fungal species, A. fumigatus and A. nidulans were the most common. Paecilomyces variotii, Rhizomucor pusillus, Thermomyces lanuginosus, Thermoascus thermophilus and Sporotrichum thermophilum were fairly common in one locality or more. The physico-chemical characteristics of water and mud samples were also followed.     

Fungal flora of poultry feedstuff ingredients

One hundred and ten samples representing five types of poultry feed ingredients were mycologically examined. These samples included soybean meal, ground maize, cotton-seed cake, wheat bran and fish meal (22 samples each). Among the 73 mesophilic fungal species, Aspergillus flavus, A. niger and A. fumigatus were the most dominant. A. terreus, A. flavipes, Mucor circinelloides, Scopulariopsis brevicaulis, Penicillium chrysogenum, Fusarium moniliforme and Rhizopus stolonifer were found to be common on a particular ingredient and less common on the remainders. Of the twelve thermophilic and thermotolerant species, A. fumigatus, Thermomyces lanuginosus, Rhizomucor pusillus and Thermoascus thermophilus prevailed on one or more type of the different ingredients. Marked variations were observed in the rancid fatty compounds of the different samples and the values ranged between 0.663 and 3.900 mg malonaldehyde per kg sample.     

Mycoflora of air-conditioners dust from Riyadh, Saudi Arabia

Using the hair baiting technique, 6 genera and 14 species were collected on Sabouraud's dextrose agar from 37 dust samples from air-conditioners. The most common fungi were Chrysosporium tropicum, C. indicum, C. keratinophilum, Aspergillus flavus followed by Acremonium strictum and Scopulariopsis brevicaulis. Using the dilution-plate method, 26 genera and 52 species were collected from 37 dust samples on glucose-(23 genera and 45 species) and cellulose-(18 genera and 34 species) Czapek's agar at 28 degrees C. The most prevalent species were Aspergillus niger, A. flavus, Penicillium chrysogenum, Stachybotrys chartarum, Ulocladium atrum, Mucor racemosus and Fusarium solani and A. niger, A. flavus, Trichoderma viride, P. chrysogenum, Ulocladium atrum, Chaetomium globosum, C. spirale, Stachybotrys chartarum and Mucor racemosus on the two media, respectively.     

Mycoflora associated with five species of freshwater leeches.

Twenty-two species belonging to 11 genera of zoosporic fungi were collected from 10 freshwater sites and from five species of leeches on sesame and hemp seeds, maize grains and Pinus pollen grains as baits at 22 degrees C. Dictyuchus, Achlya, Pythium, Allomyces and Saprolegnia were the most common genera. The highest population of zoosporic fungi was estimated from Batracobdelloides tricarinata, the lowest from Barbronia assiuti. In addition fifty-three species and 1 variety representing 23 terrestrial fungal genera were identified on glucose (17 genera and 41 species) and cellulose (20 genera and 34 species + 1 variety) Czapek's Dox agar at 28 degrees C. The most common species on the two media were Aspergillus niger, A. fumigatus, A. flavus, Trichoderma harzianum, Gibberella fujikuroi and Penicillium chrysogenum. Some fungi were common only on cellulose agar medium such as Botryotrichum atrogriseum, Chaetomium bostrychodes, Dactylella alaskana and Drechslera halodes. Batracobdelloides tricarinata was the richest with terrestrial fungi whereas Salifa delicata was the poorest. Five leech species, namely Alboglossiphonia polypompholyx, Batracobdelloides tricarinata, Helobdella conifera, Barbronia assiuti and Salifa delicata were used during this investigation.     

Occurrence of zoosporic and terrestrial fungi in some ponds of Kharga Oases, Egypt

Thirty-nine species belonging to 13 genera of zoosporic fungi were collected from 33 water samples on sesame and hemp seeds as baits at 22 degrees C. Five genera were isolated in high occurrence: Allomyces (2 species), Achlya (9 species), Dictyuchus (4 species), Pythium (5 species) and Saprolegnia (5 species). Aphanomyces (4 species) and Isoachlya (2 species) were of moderate frequencies. The remaining six genera were less frequent: Anisolpidium, Brevilegnia, Calyptralegnia, Leptolegnia, Pythiopsis and Thraustotheca. Sixty-six species and 2 varieties belonging to 34 genera were identified on glucose-(24 genera and 52 species + 1 variety) and cellulose (31 genera and 48 species + 2 varieties) Czapek Dox agar at 28 degrees C. The most common species on the two media were Acremonium strictum, Aspergillus flavus, A. fumigatus, A. niger, Fusarium moniliforme, F. oxysporum, F. solani, Mucor racemosus, Penicillium chrysogenum and P. corylophilum. Some fungi were common only on cellulose agar medium such as Chaetomium globosum, C. spirale and Memnoniella echinata.     

Mycoflora of anise and fennel seeds in Egypt

Using four medium types (glucose-, cellulose-, 50% sucrose- and 10% NaCl-Czapek's agar), it was possible to isolate 15 fungal genera, 78 species and 6 varieties. The collective fungal spectrum varied from one medium to another where the highest number of species (57 species/1000 seeds) was obtained on glucose- and the lowest (31 species/1000 seeds) on 10% NaCl-Czapek's agar. Aspergillus, Penicillium, and sometimes Rhizopus and Chaetomium were the most common genera on the different medium types. The most common fungal species especially on glucose- and cellulose-Czapek's agar were, Aspergillus niger, A. flavus, A. ochraceus, A. fumigatus, A. flavus var. columnaris, Penicillium chrysogenum, Rhizopus stolonifer and Chaetomium globosum. Members of A. glaucus group were more frequently recovered on 10% NaCl- and 50% sucrose-Czapek's agar.     

Assay of cellulolytic activity of cellulose-decomposing fungi isolated from Egyptian soils

Ninety two fungal species recovered on cellulose — Czapek's agar plates from Egyptian soils were assayed for their cellulolytic activities either at 28 °C (mesophilic) or at 45 °C (thermophilic or thermotolerant). The percentage increase of protein-nitrogen over those of control was taken as a measure of this ability. At 28°C, the top species in cellulolytic activity were Penicillium oxalicum, Nectria haematococca, Gliocladium catenulatum, Myrothecium verrucaria and Aspergillus niger. The other species showed low, weak or non cellulalytic activity. At 45°C, the highest activity was manifested by Chactomium olivaceum, Sporotrichum thermophilum, Sepedonium chrysospermum, Aspergillus fumigatus, Humicola minima, Rhizomucor pusillus and Thermomyces lanugibnosus. All Penicillium species tested as well as some other fungal species were not capable of utilizing cellulsoe at 45°C.     

Fermentation and antimicrobial activities of extracts from different species of fungus belonging to Genus, Trichoderma

The present paper discusses the effects of the crude extracts of Trichoderma spp. on a number of fungal and bacterial organisms. These include Paecilomyces variotii, Penicillium notatum, Nematospora corylii, Mucor miehei, Bacillus brevis, Bacillus subtilis, Enterobacter dissolvens and Sarcina lutea. The culture broth extracts of different isolates of Trichoderma harzianum, T. longibrachiatum and T. koningii cultured in different media were investigated individually for in-vitro antifungal and antibacterial activities by agar diffusion technique. The culture broth extracts of T. harzianum produced definite antifungal and antibacterial activities against most of the test organisms. The results indicate that the extracts were fungicidal and antibacterial at a concentration of 100 microg per 6mm paper disks. Trichoderma harzianum showed the highest activity while T. koningii showed the least activity against most of the test organisms especially the yeast Nematospora coryli. The results of this study indicate that the Trichoderma species especially T. harzianum is a possible source of useful antimicrobial agents against pathogenic microorganisms which include gram-negative, gram-positive, fungi and yeasts.     

Sensitivity of different zygomycetes to the Penicillium chrysogenum antifungal protein (PAF)

The effects of the Penicillium. chrysogenum low molecular mass antifungal protein (PAF) on selected fungal species belonging in the Zygomycetes were investigated. A total of 21 fungal isolates from 15 different genera (Absidia, Actinomucor, Cokeromyces, Gilbertella, Micromucor, Mortierella, Mucor, Mycotypha, Rhizomucor, Rhizopus, Saksenaea, Syncephalastrum, Thamnostylum, Umbelopsis and Zygorhynchus) were tested. The inhibitory potentials of PAF at 50 microg ml(-1) on the germination of the sporangiospores and at 6.25-50 microg ml(-1) on the hyphal extension were examined on different culture media. From among the fungi regarded as opportunistic human and/or animal pathogens, PAF exhibited inhibitory effects against Absidia, Mortierella, Rhizomucor and Rhizopus species.     

Saprophytic fungi isolated from animal and bird pens in Egypt

Forty-four samples collected from animal and bird pens were screened for their content of saprophytic fungi by using the dilution plate method. 76 species in addition to one variety of Aspergillus flavus belonging to 33 genera were recovered on three types of media: 20 genera and 49 species on Littman-oxgall agar, 19 genera and 41 species on cellulose- and 19 genera and 43 species on glucose-Czapek's agar. The most frequent genera were Aspergillus (21 species), Scopulariopsis (4 species) and Penicillium (10 species). Among the isolated fungi A. fumigatus, A. flavus, A. terreus, A. niger, A. sydowi, A. nidulans, S. brevicaulis, S. brumptii, P. chrysogenum and P. funiculosum were the most common species.     

The role of chlamydospores of Paracoccidioides brasiliensis.

The role of chlamydospores in the conversion process from a mycelial-to-yeast form using the slide culture method was studied. Three clinical isolates and two other isolates from armadillo, belonging to the fungal species Paracoccidioides brasiliensis, were cultured on Sabouraud dextrose agar (SDA), potato dextrose agar (PDA) and brain heart infusion dextrose agar (BHIDA). Initially, the mycelial forms of each isolate were grown at 25 degrees C for 7, 14, 30 or 60 days on slide cultures and then the temperature was shifted to 35 degrees C. Interestingly, the slide cultures of all the isolates at 25 degrees C formed chlamydospores on either SDA or BHIDA, whereas, on PDA medium, aleurioconidia were formed. If the slide cultures on BHIDA were incubated at 35 degrees C for 7 to 14 days, multiple budding forms could be observed. This phenomenon was not evident in the slide cultures of SDA or PDA. The results of this morphological study indicate that in P. brasiliensis, chlamydospores may play an important role in the conversion process from a mycelial-to-yeast form.     

The paf gene product modulates asexual development in Penicillium chrysogenum

Penicillium chrysogenum secretes a low molecular weight, cationic and cysteine-rich protein (PAF). It has growth inhibitory activity against the model organism Aspergillus nidulans and numerous zoo- and phytopathogenic fungi but shows only minimal conditional antifungal activity against the producing organism itself. In this study we provide evidence for an additional function of PAF which is distinct from the antifungal activity against putative ecologically concurrent microorganisms. Our data indicate that PAF enhances conidiation in P. chrysogenum by modulating the expression of brlA, the central regulatory gene for mitospore development. A paf deletion strain showed a significant impairment of mitospore formation which sustains our hypothesis that PAF plays an important role in balancing asexual differentiation in P. chrysogenum.     

The prevalence of IgE antibody reactivity against the alkaline serine protease major allergen of Penicillium chrysogenum increases with the age of asthmatic patients.

BACKGROUND: Penicillium species are prevalent airborne fungi. However, the prevalence of allergic sensitization to Penicillium antigens and the true impact of these ubiquitous fungi on atopic respiratory disorders remain to be determined. OBJECTIVE: The purpose of this study was to analyze the prevalence of immunoglobulin (Ig)E and IgG antibodies against Penicillium chrysogenum (Pen ch 13), the alkaline serine protease major allergen of P. chrysogenum, in asthmatic patients of different age groups. METHODS: Pen ch 13 was purified from a culture medium of P. chrysogenum. The reactivity of IgE and IgG antibodies to Pen ch 13 in the serum samples of 212 asthmatic patients was analyzed by immunoblotting methods. RESULTS: Sixty-nine (33%) of the 212 sera analyzed showed IgE and/or IgG immunoblot reactivity to Pen ch 13. Significant differences in the prevalence of IgE and/or IgG antibody reactivity to Pen ch 13 were found among eight different age groups of 212 asthmatic patients. The frequency of IgE-binding reactivity to Pen ch 13 increased significantly with the age of the patients. It was 7% for the group less than 10 years old and 42% for the group older than 70 years old. In addition, a significant difference between the prevalence of IgE (7%) and IgG (33%) antibodies against Pen ch 13 in the group aged 10 or less was also found. CONCLUSIONS: Our study demonstrates that IgE and IgG antibodies specific for Pen ch 13 were detected in approximately one-third of the 212 asthmatic patients analyzed. Our results suggest that allergic sensitization to Pen ch 13, and possibly to other airborne Penicillium species, is more common in older asthmatic patients.     

Peritonitis due to Thermoascus taitungiacus (Anamorph Paecilomyces taitungiacus)

The first case of human disease due to the thermophilic ascomycete Thermoascus taitungiacus (the teleomorph of Paecilomyces taitungiacus) is presented. T. taitungiacus was recovered from four dialysate fluid specimens of a 57-year-old patient undergoing chronic peritoneal dialysis. Identification was based upon cylindrical conidia, reddish orange nonostiolate ascomata, lack of growth at 20 degrees C, thermotolerance, and ascospores that appeared pale yellow, elliptical, thick walled, and predominately echinulate by light microscopy but irregularly verrucose by scanning electron microscopy.     

Production of lipase by soil fungi and partial characterization of lipase from a selected strain (Penicillium wortmanii)

Filamentous fungi from soil were screened for their ability to produce lipase. Among 56 filamentous fungi tested, one strain identified as Penicillium wortmanii was selected as the highest lipase producer. Maximum lipase production (12.5 U/ml) was obtained in 7-days cultures utilizing 5% (w/v) olive oil as the carbon source. Optimum pH and temperature for crude lipase were 7.0 and 45 degrees C, respectively. The enzyme was stable at 40 and 45 degrees C and it retained about 55% of its activity when heated at 50 degrees C for 1 hour.     

Esterases and putative lipases from tropical isolates of Aureobasidium pullulans

Esterases and lipases have been studied in a number of fungi, though very little is known about esterases from Aureobasidium pullulans especially from the African tropics. In this study, forty-two Zimbabwean isolates were screened for lipase activity on tributyrin agar. Extracellular esterase activities of seven selected isolates were studied under varying conditions using para-nitrophenol acetate as substrate. Twenty isolates (48%) showed lipolytic activity; sixteen showed negative results for lipase activity while the rest showed weak activities. Esterase activities in broth cultures ranged from 0.011-0.223 mmol/microg protein/min while activities ranged from 1.5-12.8 U/ml under solid state fermentation. The esterases were optimally active at pH 7.6-8.0, showed a temperature optimum of 35 degrees C and retained more than 50% activity at temperatures up to 60 degrees C and at pH 4.0-7.0 after 150 min. Enzyme production was optimal after 5-6 days with diammonium hydrogen phosphate as nitrogen source. Isolates showed variations in preference for carbon source for esterase production. The A. pullulans esterases differed from most fungal esterases in that they are optimally active in alkaline conditions and are active over a broad pH range.     

Beta-glucuronidase activities of fecal isolates from healthy swine.

Research has shown that various percentages of fecal Escherichia coli isolates obtained from healthy subjects may be beta-glucuronidase negative. The ability to detect beta-glucuronidase activity among fecal E. coli isolates from healthy subjects may be affected by assay conditions. A study was conducted in which agar and broth media containing 4-methylumbelliferyl-beta-D-glucuronide (MUG) were used to examine beta-glucuronidase activities of fecal isolates from healthy swine. Rectal swabs were plated on MacConkey agar plus 100 mg of MUG per liter (MAC-MUG) and incubated at 35 degrees C for 24 h. Each of 986 isolates picked from MAC-MUG was inoculated into duplicate tubes of lauryl tryptose broth plus 50 mg of MUG per liter (LT-MUG). One set of tubes was incubated at 35 degrees C and the other set of tubes was incubated at 44.5 degrees C. Gas production and hydrolysis of MUG, indicated by fluorescence when observed with UV light with a wavelength of 360 nm, were determined after incubation for 24 and 48 h. A higher percentage (P less than 0.01) of isolates was MUG positive at 44.5 degrees C than at 35 degrees C after 24 h of incubation in LT-MUG. A higher percentage (P less than 0.01) of isolates was MUG positive after 48 h than after 24 h of incubation at both 35 and 44.5 degrees C. A lower percentage of isolates (P less than 0.05) was observed to be MUG positive on MAC-MUG agar compared with their MUG reactions in LT-MUG at 35 and 44.5 degrees C. Approximately 89% of the isolates identified were beta-glucuronidase-positive E. coli. The largest proportion of MUG-positive E. coli was detected with LT-MUG at 35 degrees C after 48 h of incubation.     

In vitro susceptibility testing of filamentous fungi: comparison of Etest and reference microdilution methods for determining itraconazole MICs

The performance of the Etest for itraconazole susceptibility testing of 50 isolates of filamentous fungi was assessed in comparison with the National Committee for Clinical Laboratory Standards (NCCLS) proposed standard microdilution broth method. The NCCLS method employed RPMI 1640 broth medium, and MICs were read after incubation for 48 h at 35 degrees C. Etest MICs were determined with RPMI agar containing 2% glucose and with Casitone agar and were read after incubation for 24 h (Aspergillus spp. and Rhizopus spp.) and 48 h (all species except Rhizopus spp.) at 35 degrees C. The isolates included Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Aspergillus terreus, Fusarium spp., Pseudallescheria boydii, Rhizopus spp., Paecilomyces variotii, and an Acremonium sp. Overall agreement between Etest and microdilution MICs was 96% with RPMI agar and 80% with Casitone agar. The agreement was 100% for all species except Rhizopus spp. (83%) and Paecilomyces varioti (0%) with RPMI agar. When Casitone agar was used, the agreement ranged from 50% with Rhizopus spp. to 100% with Fusarium spp., P. boydii, P. varioti, and an Acremonium sp. Notably, for Aspergillus spp., the agreement between itraconazole Etest MICs read at 24 h and reference microdilution MICs read at 48 h was 100% with both RPMI and Casitone agar. Both media supported the growth of all filamentous fungi tested. Where a discrepancy was observed between Etest and the reference method, the Etest MIC was generally higher. The Etest method using RPMI agar appears to be a useful method for determining itraconazole susceptibilities of Aspergillus spp. and other filamentous fungi.