Fungi isolated from olive ecosystems and screening of their potential biotechnological use

This study investigated the fungi diversity of fresh olive (Olea europaea L.) fruits, olive paste (crushed olives) and olive pomace (solid waste) and screened and quantified enzymatic activities with biotechnological applications. Fungi were randomly isolated from olive cultivars from Castilla La Mancha region (Spain). Identification included comparison of their polymerase chain reaction (PCR) amplicons of the ITS1-5.8S-ITS2 ribosomal DNA region, followed by nucleotide sequence analysis. Fourteen different species with DNA sequences of different similarities were identified, belonging to seven different genera (Aspergillus, Penicillium, Rhizomucor, Mucor, Rhizopus, Lichtheimia and Galactomyces). Aspergillus fumigatus, followed by Galactomyces geotrichum, Penicillium commune and Rhizomucor variabilis var. regularior were the most frequent species. Specific enzyme screening was assayed on agar plates, using cellobiose, carboxymethylcellulose (CMC), polygalacturonic acid and CaCl(2)/Tween 80 as substrates for β-glucosidase, carboxymethylcellulase (CMCase), polygalacturonase and lipase, respectively. Species exhibiting the best activities were: Aspergillus fumigatus (for β-glucosidase, CMCase and lipase); Rhizopus oryzae (for β-glucosidase and lipase); Rhizomucor variabilis (for β-glucosidase, CMCase and polygalacturonase); Mucor fragilis (β-glucosidase, CMCase and lipase); Galactomyces geotrichum (for β-glucosidase, polygalacturonase and lipase) and Penicillium commune and Penicillium crustosum (for lipase). The species that had shown the best enzymatic activities were grown on hemicellulose, cellulose and pectin and some activities were quantified (xylanase, cellulase, β-glucosidase and pectinase). An isolate of A. fumigatus and one of A. niger showed the best cellulase and xylanase activities, while no species presented good pectinase and β-glucosidase activities. The selected species with potential enzymatic activities could be used for future applications of industrial interest.     

Dynamics in the microbiology of maize silage during whole‐season storage

Aims: To monitor seasonal variations in the microbiology of maize silage and to determine whether the risk of fungal spoilage varies during whole‐year storage. Methods and Results: A continuous survey of 20 maize silage stacks was conducted over a period from three to 11 months after ensiling. Filamentous fungi, yeasts and lactic acid bacteria (LAB) were enumerated at five time‐points, and cultivable species of filamentous fungi were identified. Significant differences in the numbers of filamentous fungi, yeast and LAB were detected. The highest numbers of fungi were five to seven and the lowest 11 months after ensiling, while the LAB decreased in numbers during the study. Filamentous fungi were isolated from all stacks at all time‐points. The most abundant toxigenic mould species were Penicillium roqueforti, Penicillium paneum and Aspergillus fumigatus. Conclusions: There are significant variations in the microbiology of maize silage over a whole storage season. The risk of fungal spoilage was highest 5–7 months after ensiling and lowest after 11 months. Significance and Impact of the Study: This information is valuable in the assessment of health risks connected with spoiled maize silage and may be useful in the management of maize silage stacks, when whole‐season storage is applied.     

Mycoflora of Iranian Maize Harvested in the Main Production Areas in 2000

Maize is one of the most important cereals produced in, and imported into, Iran. The incidences of seed-borne fungi were determined in Iranian maize harvested in 2000 from four major production areas with different climatic conditions, namely Fars, Khuzestan, Kermanshah and Mazandaran provinces. This is the first study to compare the mycoflora of maize in the aforementioned areas. Mycological analyses showed a predominance of Fusarium species (38.5%), followed by Aspergillus species (8.7%), Rhizopus species (4.8%), Penicillium species (4.5%), Mucor species (1.1%), and four other fungal genera. Fusarium verticillioides was the most prevalent species (83% of Fusarium isolates and 52% of the total isolations), with the highest incidence in Mazandaran (59%), a region of Iran with the highest rainfall and relative humidity, high rate of esophageal cancer (EC) and high levels of fumonisins in maize. Aspergillus flavus was the most widely recovered Aspergillus species and 38% of samples were contaminated with this potentially aflatoxigenic fungus. The incidence of A. flavus was highest in Kermanshah, the province with lowest mean minimum temperature. Penicillium species were seen in all the samples and Fars had the highest incidence, with highly significant differences when compared to the other three provinces. Diplodia species were not isolated from any of the samples examined.     

Mycotoxin-producing potential of fungi associated with qat (Catha edulis) leaves in yemen

Forty-four fungal species belonging to 20 genera were isolated from 30 samples of qat leaves. The most frequent genera wereAspergillus, Alternaria, Penicillium, andCladosporium followed byFusarium, Drechslera, Chœtomium, andMucor. The most prevalent species in above genera wereAspergillus niger, A. flavus, A fumigatus, Alternaria alternata, Penicillium chrysogenum, P. citrinum, Cladosporium cladosporioides, andFusarium verticillioides. From these fungi, 17 species (39%) related to 7 genera (35%) proved to be true endophytes. Eleven out of 75 isolates were mycotoxigenic.A. alternata produced alternariol and alternariol monomethyl ether whereasA. flavus produced aflatoxins B₁ and B₂. Ochratoxin A, sterigmatocystin, citrinin and T-2 toxin were produced byA. ochraceus, A. versicolor, P. citrinum andF. oxysporum, respectively. The presence of such toxigenic fungi associated with qat leaves is considered to be a threat to public health.     

A PCR-ELISA for the detection of potential fumonisin producing Fusarium species

A PCR-ELISA for the detection of potential fumonisin producing Fusarium species has been developed, using the ribosomal ITS1 sequence as target. For this purpose, the sequences of the ITS1 regions of different fumonisin producing Fusarium species have been determined and compared to the sequences of fumonisin non-producing species. In general, the ITS1 sequences were highly homologous. However, some minor sequence polymorphisms were detected, which differentiates potential fumonisin producing Fusarium species from non-producing species. By using these sequence differences, a PCR-ELISA for potential fumonisin producing Fusarium species was developed. All other ubiquitously occurring food-borne fungi tested showed negative results with this test.     

The mycoflora and incidence of aflatoxin,zearalenone and sterigmatocystin in dairy feed and forage samples from Eastern India and Bangladesh

The mycoflora of a range of mixed ruminant feed and forage samples collected in Eastern India and Bangladesh were determined. Most fungi isolated from the samples were those associated with low moisture contents. Several potentially mycotoxigenic fungi and thermotolerant species were present particularly in the rice straw. The most frequently isolated fungi were: Aspergillus versicolor (Vuill.) Tiraboschi, Penicillium citrinum Thorn, Eurotium species, Wallemia sebi (Fr.) v. Arx, Aspergillus penicilloides Speg. and yeasts. Fusarium moniliforme Sheldon and Cladosporium species were prevalent in straw but not other feeds. Levels of certain individual fungi, particularly thermotolerant types, were higher in Bangladesh rice straw although overall counts were similar to those of the Indian samples. Mycotoxin analyses for aflatoxins, zearalenone and sterigmatocystin were carried out. Aflatoxins were detected in significant amounts in some feed samples from Eastern India but not in rice straw from India and Bangladesh. Zearalenone occurred in a Paspalum straw sample and three other feed samples from India and sterigmatocystin was detected in three rice straw samples from Bangladesh.     

Identification of fungi associated with rotylenchulus reniformis

The objective of this work was to isolate and identify fungi associated with R. reniformis in cotton roots. Soil samples were collected in cotton fields naturally infested with R. reniformis and from cotton stock plants cultured in the greenhouse. Nematodes extracted from the soil were observed under the stereoscope, and discolored eggs and vermiform stages colonized with mycelia were cultured on 1.5% water agar supplemented with antibiotics, and incubated at 27°C. Identification of the nematophagous fungi was based on the morphological characters, and the ITS regions and 5.8S rDNA amplified by PCR using the primers ITS1 and ITS4. The parasitism percentage on vermiform nematodes from greenhouse samples was 21.2%, and the percentages from cotton fields in Limestone, Henry, and Baldwin counties in Alabama were 3%, 23.2%, and 5.6%, respectively. A total of 12 fungi were identified from R. reniformis vermiform stages and eggs. The most frequently isolated fungi were Arthrobotrys dactyloides (46%) and Paecilomyces lilacinus (14%), followed by Phoma exigua (4.8%), Penicillium waksmanii and Dactylaria brochophaga (3.6%), Aspergillus glaucus group (2.4%). Cladosporium herbarum, Cladosporium cladiosporioides, Fusarium oxysporum, Torula herbarum, Aspergillus fumigatus, and an unidentified basidiomycete were less frequent (1.2%). A high percentage (16.8%) of fungi from colonized nematodes was not cultivable on our media. Out of those 12 fungi, only four have been previously reported as nematophagous fungi: three isolates of Arthrobotrys dactyloides, and one isolate of Dactylaria brochopaga, Paecilomyces lilacinus, and Fusarium oxysporum. Molecular identification of Arthrobotrys dactyloides and Dactylaria brochopaga was consistent with the morphological identification, placing these two fungi in the new genus Drechslerella as proposed in the new Orbilaceae classification.     

Isolation,identification and testing for allergenicity of fungi from air-conditioned indoor environments

Twelve fungi were isolated and identified from air-conditioned rooms. Of the 12, 7 were species of Aspergillus, viz. A. niger, A. oryzae, A. fumigatus, A. terreus, A. nidulans, A. versicolor and A. parasiticus. Other fungi were Penicillium citrinum, Fusarium oxysporum, Trichoderma viride, Neurospora crassa and Alternaria alternata. A. niger was present in 80% of the locations. Some of the fungi isolated in this study could be opportunistic fungal pathogens, like A. fumigatus, A. niger and Penicillium citrinum, and were found to be allergenic. Results of this study indicate that air-conditioned rooms could be reservoirs of fungi and may cause allergic problems or infections in healthy or immunocompromised individuals living in these environments.     

Ecological and physiological studies on fungi associated with human hair

Thirty-seven species attributed to 19 genera of keratinophilic fungi were recovered from 100 human hair samples collected from the Assiut governorate. The genera Aspergillus followed by Penicillium and Chrysosporium were frequently isolated from 65, 43 and 30% of the samples respectively. Fifteen species and 13 genera of thermophilic and thermotolerant fungi (recovered at 45 degrees C) were identified. The thermotolerant Aspergillus fumigatus was frequently encountered and emerged from 82% of the samples. Thirteen isolates of keratinophilic and 20 isolates of thermophilic fungi were tested for lipolytic and proteolytic activities. All the keratinophilic fungi showed lipolytic and proteolytic activities while 100 and 85% of the thermophilic fungi showed lipolytic and proteolytic activities. Using the paper-disc plate method, 12 types of shampoos and oils were tested for their antifungal activities on 42 strains of keratinophilic and thermophilic or thermotolerant fungi. Three out of four types of shampoo proved to be highly effective against all the test fungi.     

Identification of the pathogenic Aspergillus species by nested PCR using a mixture of specific primers to DNA topoisomerase II gene

For PCR-based identification of Aspergillus species, a common primer of the DNA topoisomerase II genes of Candida, Aspergillus and Penicillium, and species-specific primers of the genomic sequences of DNA topoisomerase II of A. fumigatus, A. niger, A. flavus (A. oryzae), A. nidulans and A. terreus were tested for their specificities in PCR amplifications. The method consisted of amplification of the genomic DNA topoisomerase II gene by a common primer set, followed by a second PCR with a primer mix consisting of 5 species-specific primer pairs for each Aspergillus species. By using the common primer pair, a DNA fragment of approximately 1,200 bp was amplified from the Aspergillus and Penicillium genomic DNAs. Using each species-specific primer pair, unique sizes of PCR products were amplified, all of which corresponded to a species of Aspergillus even in the presence of DNAs of several fungal species. The sensitivity of A. fumigatus to the nested PCR was found to be 100 fg of DNA in the reaction mixture. In the nested PCR obtained by using the primer mix (PsIV), the specific DNA fragment of A. fumigatus was amplified from clinical specimens. These results suggest that this nested PCR method is rapid, simple and available as a tool for identification of pathogenic Aspergillus to a species level.     

Molecular identification of species from the Penicillium roqueforti group associated with spoiled animal feed

The Penicillium roqueforti group has recently been split into three species, P. roqueforti, Penicillium carneum, and Penicillium paneum, on the basis of differences in ribosomal DNA sequences and secondary metabolite profiles. We reevaluated the taxonomic identity of 52 livestock feed isolates from Sweden, previously identified by morphology as P. roqueforti, by comparing the sequences of the ribosomal internal transcribed spacer region. Identities were confirmed with random amplified polymorphic DNA analysis and secondary metabolite profiles. Of these isolates, 48 were P. roqueforti, 2 were P. paneum, and 2 were Penicillium expansum. No P. carneum isolates were found. The three species produce different mycotoxins, but no obvious relationship between mold and animal disease was detected, based on medical records. P. roqueforti appears to dominate in silage, but the ecological and toxicological importance of P. carneum and P. paneum as feed spoilage fungi is not clear. This is the first report of P. expansum in silage.     

Alternaria alternata prevalence in cereal grains and soybean seeds from Entre Ríos, Argentina

A mycological survey was carried out at Entre Ríos province, Argentina, on sorghum grain, maize, rice, soybean seeds and on freshly harvested and stored wheat. The isolation frequencies and relative densities of species belonging to genera Alternaria, Aspergillus, Fusarium, Penicillium and other fungi were calculated. Alternaria alternata was the major fungal species isolated from sorghum, rice, soybean seeds and on freshly harvested wheat, and a low incidence of Fusarium species was observed on the same substrates. In maize the major fungal species isolated was Fusarium verticillioides. The high incidence levels of A. alternata observed,suggest that it may be necessary to determine, among other mycotoxins, if Alternaria toxins occur in these commodities.     

The influence of bacterial inoculants on the microbial ecology of aerobic spoilage of barley silage.

The aerobic decomposition of barley silage treated with two inoculants (LacA and LacB) containing mixtures of Lactobacillus plantarum and Enterococcus faecium was investigated over a 28-day period. Initially, yeast and bacterial populations were larger in silage inoculated with LacA than in silage treated with LacB or water alone (control). Differences in the succession of yeasts in silage treated with LacA were observed relative to the other two treatments. From silage treatment with LacA, Issatchenkia orientalis was the most prevalent yeast taxon over all of the sample times, and the filamentous fungus Microascus brevicaulis was also frequently isolated at later sample dates (> or = 14 days). In contrast, Saccharomyces exiguus was the most prominent yeast recovered from silage treated with LacB and water alone on days 2 and 4, although it was supplanted by I. orientalis at later sample times. Successional trends of bacteria were similar for all three treatments. Lactobacillus spp. were initially the most prevalent bacteria isolated, followed by Bacillus spp. (primarily Bacillus pumilus). However, the onset of Bacillus spp. prominence was faster in LacA silage, and Klebsiella planticola was frequently recovered at later sample times (> or = 14 days). More filamentous fungi were recovered from LacA silage on media containing carboxylmethylcellulose, pectin, or xylan. The most commonly isolated taxa were Absidia sp., Aspergillus flavus, Aspergillus fumigatus, Byssochlamys nivea, Monascus ruber, Penicillium brevicompactum, Pseudoallescheria boydii, and M. brevicaulis. The results of this study indicated that the two bacterial inoculants incorporated into barley at the time of ensilage affected the microbial ecology of silage decomposition following exposure to air. However, neither of the microbial inoculants effectively delayed aerobic spoilage of barley silage, and the rate of decomposition of silage treated with one of the inoculants (LacA) was actually enhanced.     

The sequence of the isoepoxydon dehydrogenase gene of the patulin biosynthetic pathway in <Emphasis Type="Italic">Penicillium</Emphasis> species

Interest in species of the genus Penicillium is related to their ability to produce the mycotoxin patulin and to cause spoilage of fruit products worldwide. The sequence of the isoepoxydon dehydrogenase (idh) gene, a gene in the patulin biosynthetic pathway, was determined for 28 strains representing 12 different Penicillium species known to produce the mycotoxin patulin. Isolates of Penicillium carneum, Penicillium clavigerum, Penicillium concentricum, Penicillium coprobium, Penicillium dipodomyicola, Penicillium expansum, Penicillium gladioli, Penicillium glandicola, Penicillium griseofulvum, Penicillium paneum, Penicillium sclerotigenum and Penicillium vulpinum were compared. Primer pairs for DNA amplification and sequencing were designed from the P. griseofulvum idh gene (GenBank AF006680). The two introns present were removed from the nucleotide sequences, which were translated to produce the IDH sequences of the 12 species for comparison. Phylogenetic relationships among the species were determined from rDNA (ITS1, 5.8 S, ITS2 and partial sequence of 28S rDNA) and from the idh nucleotide sequences minus the two introns. Maximum parsimony analysis showed trees based on rDNA and idh sequences to be congruent. It is anticipated that the genetic information obtained in the present study will aid in the design of probes, specific for patulin biosynthetic pathway genes, to identify the presence of these mycotoxigenic fungi. The U.S. Government's right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged.     

Lactobacillus plantarum MiLAB 393 produces the antifungal cyclic dipeptides cyclo(L-Phe-L-Pro) and cyclo(L-Phe-trans-4-OH-L-Pro) and 3-phenyllactic acid

We have isolated a Lactobacillus plantarum strain (MiLAB 393) from grass silage that produces broad-spectrum antifungal compounds, active against food- and feed-borne filamentous fungi and yeasts in a dual-culture agar plate assay. Fusarium sporotrichioides and Aspergillus fumigatus were the most sensitive among the molds, and Kluyveromyces marxianus was the most sensitive yeast species. No inhibitory activity could be detected against the mold Penicillium roqueforti or the yeast Zygosaccharomyces bailii. An isolation procedure, employing a microtiter well spore germination bioassay, was devised to isolate active compounds from culture filtrate. Cell-free supernatant was fractionated on a C(18) SPE column, and the 95% aqueous acetonitrile fraction was further separated on a preparative HPLC C(18) column. Fractions active in the bioassay were then fractionated on a porous graphitic carbon column. The structures of the antifungal compounds cyclo(L-Phe-L-Pro), cyclo(L-Phe-trans-4-OH-L-Pro) and 3-phenyllactic acid (L/D isomer ratio, 9:1), were determined by nuclear magnetic resonance spectroscopy, mass spectrometry, and gas chromatography. MIC values against A. fumigatus and P. roqueforti were 20 mg ml(-1) for cyclo(L-Phe-L-Pro) and 7.5 mg ml(-1) for phenyllactic acid. Combinations of the antifungal compounds revealed weak synergistic effects. The production of the antifungal cyclic dipeptides cyclo(L-Phe-L-Pro) and cyclo(L-Phe-trans-4-OH-L-Pro) by lactic acid bacteria is reported here for the first time.     

Mycoflora and trichothecene toxins of paddy grains from Egypt

120 species and 38 genera were collected from 64 samples of paddy grains on glucose- and cellulose-Czapek's agar at 28 °C. The total count of glycophilic and cellulose-decomposing fungi fluctuated between 216–29760; and 124–11320 colonies/g paddy grains on the two media, respectively.On glucose agar, the most common species were Aspergillus niger, A. flavus, A. sydowi, A. terreus, A. fumigatus, A. ochraceus, Penicillium chrysogenum, P. corylophilum, Fusarium oxysporum, Alternaria alternata, Cladosporium cladosporioides, Trichoderma viride and Mucor racemosus.On cellulose agar with pH 5.5 & 8.0, the most prevalent fungi were Stachybotrys chartarum, S. bisybi, Aspergillus niger, A. flavus, Fusarium oxysporum, Alternaria alternata, Drechslera sativus and Acremonium strictum.Extracts from 64 paddy samples were tested against brine shrimp larvae (Artemis salina). Of these 9 displayed varying degrees of toxicity. Trichothecene-toxins were detected in the extracts of three paddy samples only. Diacetoxyscirpenol and T-2 toxin were detected in two samples and only T-2 toxin in the other.     

Aerometric study of viable fungus spores in an animal care facility.

Studies of airborn fungi were undertaken to evaluate exposure risks for laboratory animals and human handlers which might lead to allergic or invasive disease. Although sporadically high fungus levels were encountered, counts of viable fungus particles were in general low. Recoveries on malt extract agar significantly exceeded those on Sabouraud dextrose agar. The taxa most frequently and abundantly recovered were Penicillium species. Data analyses suggest that 'clean' bedding material may be the principal source of these spores, that cleaning temporarily increases spore levels, and that outdoor airborne fungi contributed little to the indoor air spora identified. Aspergillus fumigatus was infrequently encounted in our samples, and dermatophytes were not recovered.     

Fungal flora on board the Mir-Space Station, identification by morphological features and ribosomal DNA sequences

This report is on the morphological and molecular biological identification, using 18S- and ITS1-rDNA sequences, of the "space fungi" isolated on board the Russian Mir-Space Station as the major constituents of the fungal flora. The six fungal strains were isolated from air by using an air sampler or from condensation. Strains were identified as Penicillium chrysogenum, Aspergillus versicolor, or Penicillium sp. by both methods. The species of space fungi were common saprophytic fungi in our living environment, potential pathogens, and allergens. This study concluded that the environment on board the space station Mir allows the growth of potentially pathogenic fungi as true in residential areas on the earth. Therefore, to prevent infection or other health disorders caused by these fungi, easy and reliable methods should be established to survey the fungal flora in a space station.     

Antagonistic activity of a natural fungal population towards pathogenic bacteria. An in vitro study.

In the present work, we performed in vitro testing of 33 species of fungi of the subdivision Deuteromycotina isolated from water and sediment of the Kolubara River for antagonistic action towards 11 species of pathogenic bacteria. Of gram-negative bacteria, the species most sensitive to metabolic fluid of the fungi were Proteus mirabilis, Salmonella enteritidis, and Shigella sonnei, while the most resistant were Klebsiella pneumoniae and Salmonella typhimurium. Of gram-positive bacteria, the most sensitive species was Staphylococcus aureus, while the most resistant was Enterococcus faecalis. Of the tested fungi, Penicillium canescens, P. simplicissimum, P. thomii, Aspergillus fumigatus, A. ochraceus, and Fusarium culmorum exerted inhibitory action on the greatest number of species of pathogenic bacteria, while Verticillium lateritium, V. tenerum, Phoma humicola, and Botrytis cinerea had an inhibiting effect on the least number of species.     

Fungal root endophytes from natural vegetation in Mediterranean environments with special reference to Fusarium spp

Surveys (in 2002 and 2003) were performed for fungal endophytes in roots of 24 plant species growing at 12 sites (coastal and inland soils, both sandy soils and salt marshes) under either water or salt stress in the Alicante province (Southeast Spain). All plant species examined were colonized by endophytic fungi. A total of 1830 fungal isolates were obtained and identified by morphological and molecular [internal transcribed spacer (ITS) and translation elongation factor-1alpha gene region (TEF-1alpha) sequencing] techniques. One hundred and forty-two fungal species were identified, belonging to 57 genera. Sterile mycelia were assigned to 177 morphospecies. Fusarium and Phoma species were the most frequent genera, followed by Aspergillus, Alternaria and Acremonium. Fungal root endophytic communities were influenced by the soil type where their respective host plants grew, but not by location (coastal or inland sites). Fusarium oxysporum, Aspergillus fumigatus and Alternaria chlamydospora contributed most to the differences found between endophytic communities from sandy and saline soils. Host preference was found for three Fusarium species studied. Fusarium oxysporum and Fusarium solani were especially isolated from plants of the family Leguminosae, while Fusarium equiseti showed a preference for Lygeum spartum (Gramineae). In some cases, specificity could be related to intra-specific variability as shown by sequencing of the TEF-1alpha in the genus Fusarium.