Mycobiota and molecular detection of Aspergillus flavus and A. parasiticus aflatoxin contamination of Strawberry (Fragaria ananassa Duch.) fruits

Strawberry fungi were isolated from fresh fruits and juice on the two types of media (Sabouraud dextrose agar, SDA and potato-dextrose agar, PDA) at 28 °C. Nineteen fungal species belong to 12 genera were isolated from fruits and juice on both isolation media. The most common fungal genera and species were Aspergillus flavus, A. niger, Mucor racemosus, Neurospora crassa, Penicillium chrysogenum, Rhizopus stolonifer and Trichoderma harzianum. Twenty A. flavus and A. parasitics isolates were assayed for their abilities to produce aflatoxins. The concentration of aflatoxins ranged between 25.8–75.2 and 23.6–71.1 ng/ml at 350 and 365 nm, respectively. Among A. flavus and A. parasiticus strains tested, aflatoxin B contributed 30–60% of total isolates. However, G type contributed 85–90%. The R_f values of B₁, B₂, G₁ and G₂ were 0.79, 0.61, 0.44 and 0.32, respectively. High-performance liquid chromatography analysis of extracts revealed the presence of aflatoxins with variable levels.     

Evaluation of the mycological status of luncheon meat with special reference to aflatoxigenic moulds and aflatoxin residues

The luncheon meat samples analyzed, which were produced locally by the two main luncheon meat producing companies in Egypt were relatively highly contaminated either by moulds and yeasts in general, aflatoxigenic species and aflatoxin residues in particular. The most frequently encountered fungi from the samples were yeasts, Aspergillus niger, A. flavus, Penicillium chrysogenum, Rhizopus stolonifer, Mucor circinelloides. Less common were Cladosporium sphaerospermum, Alternaria alternata, Mycosphaerella tassiana, P. aurantiogriseum and P. oxalicum. The most important aflatoxigenic species, A. flavus, was isolated frequently. It was 10% of the total fungal isolates from both samples of the two companies. Seven luncheon meat samples out of 50 analyzed were positive for aflatoxin B₁ or B₁ and G₁, while all samples were negative for aflatoxins B₂, G₂, M₁ and M₂. Aflatoxin B₁ was detected only in 4 and 3 samples out of 25 analyzed from each of company A and B, respectively. The highest detectable level, 11.1 ppb, was recorded in a sample from company B and the least, 0.5 ppb, in a sample from company A. Aflatoxin G₁, at concentration of 3.2 ppb, was detected in only one sample of the aflatoxin B₁ – contaminated 3 samples of company B: this sample also had the highest level of aflatoxin B₁. Some luncheon meat samples had higher numbers of aflatoxigenic A. flavus than others, however these samples were negative for aflatoxins. The hazardous potential of such contamination will be discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Immunoassay identification ofAspergillus flavus using monoclonal antibodies raised to the whole cell extracts

Five separate monoclonal antibodies were produced against whole cell extracts ofAspergillus flavus and ELISA procedures used to characterise the reactivity of the antibodies to various fungal extracts. All five antibodies were specific to the aflatoxin producing fungi,A, flavus andA. parasiticus, and indicated no cross reactivity with otherAspergillus species, genera of several fungi or with other components which may be found in food samples whereA. flavus may be found.     

Penicillium siamensis sp. nov., from Thailand soil

Fungal flora of 20 samples of lentil seeds collected from Assiut governorate, Egypt, were studied. Seventeen genera and 13 species were isolated on glucose- (15 genera and 27 species), cellulose-(15 genera and 25 species)Czapek's agar media at 28 °C. The most common species were as follows: on glucose-Czapek's agar, Aspergillus fumigatus, A. niger, A. flavus, A. terreus, Penicillium notatum and Rhizopus oryzae and on cellulose agar, A. fumigatus, A. niger, A. flavus and P. notatum. Thin layer chromatographic (TLC) analysis and a biological test (Artemia salina) indicated the presence and the toxicity of aflatoxin in the extract of one sample (aflatoxins B₁, B₂, G₁ and G₂, at 20 mg/kg, total). This is the first report of the natural occurrence of aflatoxins in lentils.     

The Alkaloids of Fungi

Examination of different species of local fungi, grown on two nutritive solutions of different composition, for alkaloid formation was investigated. The formation of alkaloids was confined to four species, namely: Geotrichum candidum, Mucor hiemalis, Aspergillus flavus and Rhizopus nigricans. A comparative study of the growth as well as the formation of alkaloids by these species and by Claviceps purpurea NRRL was carried out. Methods were also described with which the different alkaloids produced by the experimental strains were identified. Peptides as well as clavine type alkaloids were detected in all cases except with Mucor hiemalis where a compound corresponding to ergosine was the only alkaloid present.     

Study of airborne fungus spores by viable and non-viable methods in Havana,Cuba

The Havana aeromycobiota diversity was studied from November 2010 to October 2011 using two complementary volumetric methods. A total of 35 fungal genera were characterised, 26 of them were recognised only by non-viable methods, six with viable methodology and the other three with both sampling methods. Furthermore, 47 species were identified by cultivation and the spores collected with the non-viable methodology. These could not be included in a specific genus, and thus, were categorised into five fungal types. In general, the main, spread worldwide, mitosporic fungi also predominated the Havana atmosphere. The predominant species were Cladosporium cladosporioides, Aspergillus flavus and Penicillium citrinum. Moreover, several Zygomycetes (Syncephalastrum racemosum, Rhizopus stolonifer and Rhizopus oryzae), Ascomycetes (Chaetomium globosum) and Basidiomycetes such as Coprinus or Ganoderma were isolated. In the present paper, the review of the airborne fungi conducted in previous studies in Cuba was completed by the detection of two new genera and the first isolation of ten new records in the Cuban atmosphere. Most of the fungi detected showed a diurnal pattern with high spore peak at 11.00–12.00.     

Rhizosphere and phyllosphere fungi of four fern plants growing in Saudi Arabia

Seventy-eight species in addition to 1 variety of A. flavus belonging to 31 genera were collected from the rhizosphere (55 species + 1 variety and 24 genera) and phyllosphere (59 species and 22 genera) of Adiantum capillus-veneris, Ceterach officinarum, Asplenium filare and Cheilanthes catanensis.In the rhizosphere, the most common genera were Aspergillus, Penicillium, Fusarium, Mucor and yeasts. In the phyllosphere, the most prevalent fungi were Cladosporium herbarum, C. sphaerospermum, Alternaria alternata, Drechslera spicifera, Aspergillus spp., Penicillium spp., Cephalosporium roseo-griseum, Myrothecium verrucaria, Mucor racemosus and yeasts.     

Cellulose-decomposing fungi of salt marshes in Egypt

Seventy-five species and three varieties which belong to thirty-four genera were identified from 74 soil samples collected from salt marshes in Egypt. The most frequent fungi wereAspergillus fumigatus, Aspergillus niger, Cladosporium herbarum andAlternatia alternata, followed byAspergillus terreus,Curvularia spicifera andPenicillium notatum. Six genera were of moderate occurrence:Penicillium, Futarium, Curvularia, Rhizopus, Stachybotrys, andChaetomium. Five genera were of low occurrence:Paecilomyces, Oephalosporium, Epicoccum, Mucor andMyrothecium.     

Composition of the fungal flora of four cereal grains in Saudi Arabia

Using the grain-plate method and on glucose-Czapek's agar at 28°C, fifty-eight species belonging to 26 genera were collected from barley (42 species and 19 genera), maize (29 species and 16 genera), sorghum (32 species and 17 genera) and wheat grains (42 species and 18 genera).The most frequent genera were Aspergillus, Penicillium, Rhizopus, Fusarium, and Mucor followed by Alternaria, Drechslera, and Curvularia. From the preceding genera Aspergillus flavus, A. niger, Penicillium notatum, Rhizopus stolonifer, Fusarium moniliforme, Mucor racemosus, Alternaria alternata, Drechslera spicifera, and Curvularia lunata were the most prevalent species in the four types of grains tested.     

Amino acid supplementation reveals differential regulation of aflatoxin biosynthesis in <Emphasis Type="Italic">Aspergillus flavus</Emphasis> NRRL 3357 and <Emphasis Type="Italic">Aspergillus parasiticus</Emphasis> SRRC 143

Aflatoxins are toxic and carcinogenic secondary metabolites produced by the fungi Aspergillus flavus and Aspergillus parasiticus. To better understand the molecular mechanisms that regulate aflatoxin production, the biosynthesis of the toxin in A. flavus and A. parasticus grown in yeast extract sucrose media supplemented with 50 mM tryptophan (Trp) were examined. Aspergillus flavus grown in the presence of 50 mM tryptophan was found to have significantly reduced aflatoxin B₁ and B₂ biosynthesis, while A. parasiticus cultures had significantly increased B₁ and G₁ biosynthesis. Microarray analysis of RNA extracted from fungi grown under these conditions revealed 77 genes that are expressed significantly different between A. flavus and A. parasiticus, including the aflatoxin biosynthetic genes aflD (nor-1), aflE (norA), and aflO (omtB). It is clear that the regulatory mechanisms of aflatoxin biosynthesis in response to Trp in A. flavus and A. parasiticus are different. These candidate genes may serve as regulatory factors of aflatoxin biosynthesis.     

Fungi associated with food and feed commodities from Ecuador

Freshly harvested soybean, rice and corn from farms and corn-based pelleted feeds were collected from ranches from the coastal and mountain regions in Ecuador during 1998, and assessed for fungal contamination. The most prevalent fungi on pelleted feed were Aspergillus flavus and Fusarium graminearum. The prevalent fungi recovered from soybean were F. verticillioides, F. semitectum, Aspergillus flavus and A. ochraceus. In rice, F. oxysporum was the most prevalent toxigenic fungal species recorded, followed by F. verticillioides and A. flavus. In corn, F. verticillioides was the most prevalent fungus isolated in both the coastal and mountain regions, with high isolation frequencies of A. flavus and A. parasiticus at the coast. Based on the toxigenic species recovered, ochratoxin A may pose a contamination risk for soybean. A higher probability of aflatoxin contamination of corn was found in the coastal samples compared to those of the mountain region, while a risk of fumonisin contamination of corn exists in both regions.This revised version was published online in October 2005 with corrections to the Cover Date.     

Study on the mycoflora of Egyptian soils

Fifty genera and 155 species, in addition to three varieties of Aspergillus nidulans, were isolated from 100 soil samples collected from different localities in Egypt. Aspergillus (33 species in addition to 3 varieties of A. nidulans), Penicillium (46 species), Fusarium (6 species) and Mucor (4 species) were of high occurrence, from which A. niger, A. fumigatus, A. terreus, M. racemosus, P. notatum, P. chrysogenum and A. flavus were dominant. Five genera were of moderate occurrence and these were Humicola, Myrothecium, Rhizopus, Cochliobolus and Alternaria. Fourteen genera were of low occurrence namely, Cunninghamella, Chaetomium, Stachybotrys, Cladosporium, Syncephalastrum, Paecilomyces, Trichoderma, Scolecobasidium, Circinella, Curvularia, Ulocladium, Botryotrichum, Sepedonium and Gliocladium. Twenty-seven genera were of rare occurrence.Fourteen species were of moderate occurrence and these were A. sydowii, M. verrucaria, P. funiculosum, A. versicolor, H. grisea, F. oxysporum, A. nidulans, F. moniliforme, A. alternata, F. solani, M. hiemalis, A. candidus, R. nigricans and A. ochraceus. The remaining species were of low or rare occurrence in soils tested.Comparison between the present results and those of others showed that there is no fungal flora characteristic cultivated soils.     

Studies on the mycoflora associated with sugarcane factory waste and pollution of River Nile in upper Egypt

Sixty-nine species and four varieties that belong to 28 genera of terrestial fungi were recovered from polluted and non-polluted water and mud samples on glucose and cellulose-Czapek's agar at 28°C. The most common species from the two substrates on the two types of media were Aspergillus flavus, A. fumigatus, A.niger, Cladosporium cladosporioides, Fusarium oxysporum, Mycosphaerella tassiana and Penicillium chrysogenum. Twenty-six species belonging to 14 genera were isolated from polluted (26 species and 14 genera) and non-polluted (17species and 10 genera) mud samples on Sabouraud's dextrose agar at 28°C. The most prevalent species were Acremonium retiulum, Alternaria alternata, Aspergillus flavus, Aphanoascus fulvescens, A. terreus, Aphanoascus sp., Penicillium funiculosum and Stachybotrys chartarum.     

Air-borne fungi in the air of Barcelona (Spain). IV. Various isolated genera

During a two-year survey on the air-borne fungi in the atmosphere of Barcelona (Spain), the following genera were isolated in decreasing order: Aureobasidium, Rhizopus, Mucor, Arthrinium, Phoma, Fusarium, Trichoderma, and Botrytis.     

Preharvest aflatoxin contamination of maize inoculated withAspergillus flavus andFusarium moniliforme

A two-year factorial experiment was utilized to test plants field-inoculated singly and in combination withAspergillus flavus andFusarium moniliforme. Pinbar inoculations were made through the husks with conidial suspensions, and 10-ear maize samples were harvested at 60 days post-silking for aflatoxin determinations. When ears were inoculated with both fungi simultaneously,F. moniliforme reduced aflatoxin formation byA. flavus isolate NRRL 3357 by approximately two-thirds.F. moniliforme had no significant effect on naturally occurring aflatoxin contamination byA. flavus. This may be due to the timing of infection by both fungi in the field. In nature,A. flavus andF. moniliforme respond differently to the environment, offering one explanation of whyF. moniliforme did not measurably affect the other fungus.     

Risk assessment for aflatoxins in foodstuffs

There had been a growing body of circumstantial evidence that aflatoxin B₁ is carcinogenic, as well as acutely toxic, to humans, but in 1987 that International Agency for Research on Cancer (IARC) of the World Health Organization (WHO) accepted that aflatoxin should be classified as a Group 1 carcinogen. Regulatory levels set by the governments of many countries are based on the premise that aflatoxin is indeed carcinogenic and the European Community agreed on 16 July 1998 a limit of 2 μg kg⁻¹ for aflatoxin B₁ in a range of foods for human consumption. The need for a risk assessment arises from the widespread occurrence of the aflatoxins in commodities resulting from contamination in storage and in the field by the fungi which produce it—Aspergillus flavus, A. parasiticus and A. nomius. Risk assessment requires both a knowledge of exposure and a detailed knowledge of the toxicology of the aflatoxins. The toxicology of aflatoxin B₁ involves a complex sequence of metabolic alterations of the molecule by enzymes in the animal tissues (usually the liver). This complexity is reflected in the very diverse range of responses by different animal species and it is likely that there will also be differences in response amongst different races of humans, and indeed even amongst different individuals of the same race.     

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.     

Patulin and aflatoxin in brown rot lesion of apple fruits and their regulation

Aspergillus flavus, A. niger, Penicillium expansum and Rhizopus stolonifer were the most frequently isolated fungi from healthy apple fruits. Alternaria alternata was the most common organism of rotten apple fruits, followed by A. niger, A. flavus, P. expansum and R. stolonifer. The prevalent type of decay, brown rot lesion, is caused by R. stolonifer followed by A. flavus, A. niger, A. alternata and P. expansum. Sodium hypochlorite had good curative properties against fruit rots. The main natural mycotoxins produced in rotten apple were patulin and aflatoxins. The optimum temperature for patulin production by P. expansum was 15 °C after 15 days. Complete inhibition of patulin formation was attained using 0.2% lemon oil and > 90% inhibition using 0.05% lemon and 0.2% orange oils. Also significant inhibition (> 90%) of aflatoxin production was observed with 0.2% lemon oil. This revised version was published online in July 2006 with corrections to the Cover Date.     

Environmental and developmental factors influencing aflatoxin production by <Emphasis Type="Italic">Aspergillus flavus</Emphasis> and <Emphasis Type="Italic">Aspergillus parasiticus</Emphasis>

Aflatoxins are carcinogenic mycotoxins formed by a number of fungi in the genus Aspergillus. The major fungi responsible for aflatoxin formation in crop seeds in the field and in storage are Aspergillus flavus and A. parasiticus. This review emphasizes developmental, environmental, biological, and chemical factors that influence aflatoxin formation by A. flavus and A. parasiticus.     

Isolation of Bacterial Antagonists of Aspergillus flavus from Almonds

Bacteria were isolated from California almond orchard samples to evaluate their potential antifungal activity against aflatoxin-producing Aspergillus flavus. Fungal populations from the same samples were examined to determine the incidence of aflatoxigenic Aspergillus species. Antagonistic activities of the isolated bacterial strains were screened against a nonaflatoxigenic nor mutant of A. flavus, which accumulates the pigmented aflatoxin precursor norsolorinic acid (NOR) under conditions conducive to aflatoxin production. Using solid and liquid media in coculture assays, 171 bacteria isolated from almond flowers, immature nut fruits, and mature nut fruits showed inhibition of A. flavus growth and/or inhibition of NOR accumulation. Bacterial isolates were further characterized for production of extracellular enzymes capable of hydrolyzing chitin or yeast cell walls. Molecular and physiological identification of the bacterial strains indicated that the predominant genera isolated were Bacillus, Pseudomonas, Ralstonia, and Burkholderia, as well as several plant-associated enteric and nonenteric bacteria. A set of 20 isolates was selected for further study based on their species identification, antifungal phenotypes, and extracellular enzyme production. Quantitative assays using these isolates in liquid coculture with a wild-type, aflatoxin-producing A. flavus strain showed that a number of strains completely inhibited fungal growth in three different media. These results indicate the potential for development of bacterial antagonists as biological control agents against aflatoxigenic aspergilli on almonds.