Taxonomy and significance of black aspergilli

Members of Aspergillus section Nigri (formerly A. niger group) are distributed worldwide and are regarded as common food spoilage fungi. Some of them are widely used and studied for industrial purposes. They are common sources of extracellular enzymes and organic acids to be used in food processing and are also used in the production of traditional foods, especially in the Orient. Products produced by strains of Aspergillus niger hold the GRAS (Generally Recognised As Safe) status from the FDA. However some species in Aspergillus section Nigri can produce ochratoxin A, a nephrotoxic mycotoxin. In spite of their industrial importance, the taxonomy of black aspergilli ( Aspergillus section Nigri ) is not clear and many attempts have been made in order to find suitable taxonomic criteria. The aim of this paper is to provide an overview of the significance of black aspergilli focusing on all the approaches made in the taxonomy of this group of fungi. Some species, such as A. carbonarius and uniseriate species can be easily recognised. In the A. niger aggregate, although speciation at molecular level has been proposed, no morphological differences can be observed and species identification will therefore remain problematic. Phylogenetic analyses of ITS and 5.8S rDNA gene region of representative black Aspergillus species and a simple key to the most common species that can be easily distinguished by morphological criteria are also included.     

Mycotoxin producing fungi

Mycotoxins are relatively small molecules characterized by a diversity of chemical structure and a diversity of biological activity. They are often genotypically specific for a group of species, but the same compound can also be formed by fungi belonging to different genera. Most of the mycotoxins known have been recognized as metabolic products of Aspergillus, Penicillium and Fusarium species. This review will be focused on aflatoxins, ochratoxins and fumonisins because of their hazard to animal and human health. The production of these mycotoxins have been usually associated with a small number of species but some recent studies have reported the production of these mycotoxins by some other species. These results show that mycotoxin production is broader than is normally thought, so the possibility can not be ruled out that new species may be a new source of unexpected mycotoxins in their natural substrates.     

Mycotoxin production by Aspergillus niger aggregate strains isolated from harvested maize in three Portuguese regions

BackgroundMaize is considered one of the crops more susceptible to mycotoxins in the world. Two of the mycotoxins commonly associated with maize are fumonisins and ochratoxin A. Aspergillus niger is a known producer of ochratoxin A and is easily found in maize. Recently, however, A. niger has been reported to produce as well fumonisins, mainly fumonisin B₂.AimsThe aim of this study was to isolate A. niger strains from maize samples collected in three Portuguese maize growing regions and to detect the production of both fumonisin B₂ and ochratoxin A.MethodsNinety five maize samples were collected, plated, and all observable Aspergillus section Nigri strains were isolated. Strains were morphologically characterized and mycotoxin production was determined by HPLC-FD.ResultsIsolations resulted in a total of 270 strains of black Aspergillus from 73 samples (77% of the samples). About 14% of those strains were found to produce ochratoxin A and 39% of the strains were found to produce fumonisin B₂.ConclusionsAn association between the production of these two mycotoxins could not be found and no conclusions could be taken whether the presence of A. niger aggregate strains will increase the risk of maize contamination with fumonisins and more specifically with fumonisin B₂.     

Aspergillus niger contains the cryptic phylogenetic species A. awamori

Aspergillus section Nigri is an important group of species for food and medical mycology, and biotechnology. The Aspergillus niger 'aggregate' represents its most complicated taxonomic subgroup containing eight morphologically indistinguishable taxa: A. niger, Aspergillus tubingensis, Aspergillus acidus, Aspergillus brasiliensis, Aspergillus costaricaensis, Aspergillus lacticoffeatus, Aspergillus piperis, and Aspergillus vadensis. Aspergillus awamori, first described by Nakazawa, has been compared taxonomically with other black aspergilli and recently it has been treated as a synonym of A. niger. Phylogenetic analyses of sequences generated from portions of three genes coding for the proteins β-tubulin (benA), calmodulin (CaM), and the translation elongation factor-1 alpha (TEF-1α) of a population of A. niger strains isolated from grapes in Europe revealed the presence of a cryptic phylogenetic species within this population, A. awamori. Morphological, physiological, ecological and chemical data overlap occurred between A. niger and the cryptic A. awamori, however the splitting of these two species was also supported by AFLP analysis of the full genome. Isolates in both phylospecies can produce the mycotoxins ochratoxin A and fumonisin B?, and they also share the production of pyranonigrin A, tensidol B, funalenone, malformins, and naphtho-γ-pyrones. In addition, sequence analysis of four putative A. awamori strains from Japan, used in the koji industrial fermentation, revealed that none of these strains belong to the A. awamori phylospecies.     

A review of mycotoxin biosynthetic pathways: associated genes and their expressions under the influence of climatic factors

Fungal contamination of agricultural commodities, particularly by mycotoxigenic fungi, represents an enormous concern for global food security in terms of feeding the world's growing population with sufficient and safe food. Not only do they reduce crop yield and quality, but they also produce substantial numbers of mycotoxins, which pose serious adverse health effects in human and animals. As the genome of most mycotoxigenic species have been sequenced, the gene clusters involved in the biosynthesis of agriculturally important mycotoxins including aflatoxins, fumonisins, ochratoxins, zearalenone and trichothecenes, have been largely identified and characterised, with their roles elucidated by researchers. This review provides a comprehensive overview of the current knowledge of genes involved in the biosynthetic pathways of mycotoxins. In addition, the influence of climatic factors including water, temperature and carbon dioxide on differential mycotoxin gene expressions have been highlighted. Overall, the relationship between the relative expression of key regulatory and structural genes under different environmental conditions is significantly correlated with mycotoxins production. This indicates that mycotoxin gene induction can be used as a reliable indicator or marker to monitor mycotoxin production pre-and-post harvest. Furthermore, current strategies to manage mycotoxin risks still require improvement. Thus, an accurate understanding of the molecular mechanisms of mycotoxin biosynthesis in mycotoxigenic species could help to develop an innovative, robust targeted control strategy. This could include the exploitation of novel compounds, which can inhibit biosynthetic genes, to minimise mycotoxin risks.     

Citric acid production by Aspergillus niger on wet corn distillers grains

AIMS: To determine which citric acid-producing strain of Aspergillus niger utilized wet corn distillers grains most effectively to produce citric acid. METHODS AND RESULTS: Citric acid and biomass production by the fungal strains were analysed on the untreated grains or autoclaved grains using an enzyme assay and a gravimetric method respectively. Fungal citric acid production on the grains was found to occur on the untreated or autoclaved grains. The highest citric acid level on the grains was produced by A. niger ATCC 9142. The autoclaved grains supported less citric acid production by the majority of strains screened. Biomass production by the fungal strains on the untreated or autoclaved grains was quite similar. The highest citric acid yields for A. niger ATCC 9142, ATCC 10577, ATCC 11414, ATCC 12846 and ATCC 26550 were found on the untreated grains. Treatment of the grains had little effect on citric acid yields based on reducing sugars consumed by A. niger ATCC 9029 and ATCC 201122. CONCLUSIONS: It is feasible for citric acid-producing strains of A. niger to excrete citric acid on wet corn distillers grains whether the grains are treated or untreated. The most effective citric acid-producing strain of A. niger was ATCC 9142. SIGNIFICANCE AND IMPACT OF THE STUDY: The study shows that the ethanol processing co-product wet corn distillers grains could be utilized as a substrate for the commercial production of citric acid by A. niger without treatment of the grains.     

Mycoflora and ochratoxin-producing strains of Aspergillus section Nigri in wine grapes in Argentina

AIMS: The aims of this work were to evaluate the mycoflora and to identify the species of Aspergillus with the potential to produce ochratoxin A (OA) from different wine grape varieties from Mendoza, Argentina. Likewise, the capacity to produce OA by Aspergillus section Nigri was studied. METHODS AND RESULTS: Fifty samples of wine grapes were obtained from a winery of Mendoza province, Argentina. The surface-disinfection method was used for mycoflora determination using the medium dichloran 18% glycerol agar (DG18). Alternaria, Aspergillus and Penicillium were identified at species level. OA production was tested in 63 strains belonging to section Nigri. Alternaria genus was the most frequent (80% of the samples) followed by Aspergillus (70%). Alternaria alternata was the only specie identified from the Alternaria genus, followed by A. niger var. niger, A. flavus among others. From Penicillium genus, P. crysogenum was the most frequent specie. From 63 strains of Aspergillus section Nigri, 41.3% were OA producers. The levels of produced toxin ranged from 2 to 24.5 ng ml-1 of culture medium. CONCLUSIONS: The presence of ochratoxigenic strains of Nigri section in this substrate suggests that they may be an important source of OA in grapes from tropical and subtropical zones. Therefore, the industry should work further to diminish the growth of these fungi and mycotoxins formation in grapes, with the aim to reduce OA content in wine products. SIGNIFICANCE AND IMPACT OF THE STUDY: The wine grape contamination with A. alternata and Aspergillus section Nigri was significant.     

Mycotoxin contamination of food in Europe: Early detection and prevention strategies

This paper reviews the early detection and prevention strategies which have been employed in Europe for the control of mycotoxin contamination of food in the context of a hazard analysis critical control point (HACCP) framework. The critical control points (CCPs) in the whole food chain where mycotoxins such as trichothecenes and ochratoxins are important have been identified. Ecological studies on the effect of environmental factors which are marginal for growth and mycotoxin production have been identified for Fusarium culmorum and F. graminearum (deoxynivlenol production), and for Penicillium verrucosum and Aspergillus ochraceus (ochratoxin production) in relation to cereal production and for A. carbonarius in relation to grapes and wine production (ochratoxin formation). To minimise the entry of these mycotoxins into the food chain, effective and rapid diagnostic tools are required to monitor the CCPs effectively. To this end the potential use of molecular imprinted polymers, lateral flow devices and molecular-based techniques for the rapid detection and quantification of the mycotoxigenic moulds or their toxins have also been developed.     

Mycotoxin-Producing Potential of Mold Flora of Dried Beans

To evaluate the potential for mycotoxin production by molds in dried beans, the mold flora of 114 samples was determined both before and after surface disinfection of the beans with 5% NaOCl. Surface disinfection substantially reduced mold incidence, indicating that contamination was mainly on the surface. The flora, both before and after disinfection, was dominated by species of the Aspergillus glaucus group, the toxicogenic species A ochracues, Penicillium cyclopium, and P. viridicatum, and species of Alternaria, Cladosporium, and Fusarium. The toxicogenic species Aspergillus flavis, A. versicolor, Penicillium Citrinum, P. expansum, P. islandicum, and P. urticae were encountered less frequently. Of 209 species of Aspergillus and Penicillium screened for mycotoxin production on sterile rice substrate, 114 produced one or more of the following mycotoxins: A. flavus, aflatoxins; A. ochraceus, ochratoxins; A. nidulans, A. unguis, and A. versicolor, sterigmatocystin; P. cyclopium, penicillic acid; P. citrinum and P. viridicatum, citrinin; P. urticae, patulin and griseofulvin. Sterigmatocystin production by A. unguis is reported for the first time.     

Comparative study of Aspergillus mycotoxin production on enriched media and construction material

Isolates of Aspergillus flavus and Aspergillus fumigatus from indoor air were compared with a known mycotoxin producer for their capacity to produce mycotoxins on a variety of enrichment media and with growth on indoor substrates such as ceiling tile and wall board. In enrichment media, four of seven isolates of A. flavus produced at least one aflatoxin and both isolates of A. fumigatus produced mycotoxins. The spectrum of mycotoxins and their concentrations varied with the strain and medium. When the mycotoxin-positive strains were grown to a dense concentration on indoor construction and finishing materials such as ceiling tile and wall boards, mycotoxins were not detected in extracts of the materials. Colonization of indoor surfaces by mycotoxin-producing strains of A. flavus and A. fumigatus may not necessarily expose inhabitants to mycotoxins or result in production of mycotoxins. Received 09 February 1999/ Accepted in revised form 28 June 1999     

Production of fungal biomass protein using microfungi from winery wastewater treatment

This study was carried out to investigate the production of fungal biomass protein (FBP) in treatment of winery wastewater using microfungi. Three fungal strains, Trichoderma viride WEBL0702, Aspergillus niger WEBL0901 and Aspergillus oryzae WEBL0401, were selected in terms of microbial capability for FBP production and COD reduction. T. viride appeared to be the best strain for FBP production due to high productivity and less nitrogen requirement. More than 5 g/L of fungal biomass was produced in shake fermentation using T. viride without nitrogen addition, and by A. oryzae and A. niger with addition of 0.5-1.0 g/L (NH4)2SO4. The FBP production process corresponded to 84-90% COD reduction of winery wastewater. Fungal biomass contained approximately 36% protein produced by two Aspergillus strains, while biomass produced by T. viride consisted of 19.8% protein. Kinetic study indicated that maximum fungal cell growth could be achieved in 24h for T. viride and 48 h for A. oryzae and A. niger. Current results indicated that it could be feasible to develop a biotechnological treatment process integrated with FBP production from the winery waste streams.     

Enumeration and identification of Aspergillus group and Penicillium species in poultry feeds from Argentina

A total of 180 samples of poultry feeds were collected during 1996 and 1997 from different factories in the south of the province of Córdoba-Argentina. They were examined for the occurrence of Penicillium spp. and Aspergillus group species. Likewise, the capacity to produce aflatoxins by the Aspergillus section flavi group was determined. The predominant species of Aspergillus were A. flavus and A. parasiticus. For Penicillium spp., P. brevicompactum, P. purpurogenum and P. oxalicum were identified. Less frequently isolated were A. candidus, A. fumigatus, A. niger, A. orizae, A. parvulus, A. tamarii, A. terreus, and P. expansum, P. funiculosum, P. minioluteum, P. pinophylum, P. restrictum, P. variabile and others. The mean value counts ranged from 1 × 103 to 9.5 × 104 CFU/g for the Aspergillus spp. and from 1.2 × 103 to 2.5 × 105 CFU/g for the Penicillium spp. When cultured on autoclaved rice kernels for 1 week in the dark at 25°C, mycotoxin production by strains of A. flavus was as follows: 21 of the 45 assayed strains (47%) produced aflatoxins. From them, 24% of the isolates produced AFB1 and AFB2 with levels from 181 to 14 545 and 6 to 3640 μg/kg respectively. Only 10 strains produced AFB1 with levels from 10 to 920 μg/kg. Fifty percent of the A. parasiticus strain was toxicogenic; six aflatoxicogenic profiles were identified. Only 10% of the strains produced all of the aflatoxins. These results showed that a potential exists for the production of mycotoxins by the Aspergillus section flavi and the Penicillium spp. They also suggested an association of mycotoxicosis with poultry feeds in Argentina. This revised version was published online in June 2006 with corrections to the Cover Date.     

Production of Ochratoxins A and B on Country Cured Ham

Two strains of Aspergillus ochraceus and six of Penicillium viridicatum isolated from country cured hams were screened for production of ochratoxins A and B. None of the isolated P. viridicatum strains yielded detectable amounts of ochratoxin A or B, whereas both strains of A. ochraceus produced ochratoxins A and B on rice, defatted peanut meal, and country cured ham. After 21 days of incubation on ham, one-third of the toxin was found in the mycelial mat on the ham surface, whereas two-thirds had penetrated into the meat to a distance of 0.5 cm.     

Increases in gene-targeting frequencies due to disruption of kueA as a ku80 homolog in citric acid-producing Aspergillus niger

Low efficiencies of gene targeting hamper functional genomics in industrially important strains of Aspergillus niger. To generate strains showing high gene-targeting frequencies in A. niger WU-2223L producing citric acid, disruption of kueA encoding Ku80 homolog was performed. Disruption of kueA increased gene-targeting frequencies to 70%, and had no effect on citric acid production.     

Putative mycotoxin-producing fungi isolated from alpataco (Prosopis flexuosa) fruits

Fungi contaminant of alpataco (Prosopis flexuosa) fruits from La Pampa province (Argentina) were identified. Alternaria alternata and Sphaeropsis sapinea were the dominant species. Phoma sp., Nigrospora sp., Preussia minima, Cladosporium sp., Pithomyces chartarum, Epicoccum nigrum, Aspergillus niger and Aspergillus speluneus were also isolated but with less frequency. Twelve strains of Alternaria alternata, the toxigenic species with higher incidence, were screened for alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TA) production. Since one isolate was able to produce AME, six isolates produced AOH and AME and two isolates produced AOH, AME and TA, these results indicate a potential risk of contamination with Alternaria toxins in this substrate.     

Aspergillus ibericus: a new species of section Nigri isolated from grapes

As part of a study on the ochratoxin producing mycoflora of grapes, several Aspergillus strains were isolated and tested for their ochratoxin A (OTA) producing abilities. Aspergillus strains of the section Nigri, which did not produce detectable amounts of OTA but which had a similar morphology to A. carbonarius, were isolated from wine grapes and/or dried vine fruit in Portugal and Spain. These strains, however, have characters that allow morphological distinction from the other species in the section, particularly the conidia size (5-7 microm), which allows separation of the species from the two most common biseriate species in section Nigri: A. carbonarius (7-9 microm) and A. niger and its aggregate species (3-5 microm). The strains are described here as belonging to a new species, named A. ibericus. The validation of this new taxon is supported further by analysis of the ITS-5.8S rDNA and calmodulin gene sequences and by analysis of the amplified fragment length polymorphism (AFLP) patterns, which were consistent in separating these strains from other species in the section. A. ibericus strains do not produce OTA therefore they are interesting for biotechnological exploration because many metabolites with commercial value are produced by other species in the section.     

Duckling toxicity and the production of fumonisin and moniliformin by isolates in the A and E mating populations of Gibberella fujikuroi (Fusarium moniliforme).

Two biological species of Gibberella fujikuroi (A and F mating populations) share the Fusarium moniliforme anamorph. Twenty strains of each of these biological species were tested for the ability to produce fumonisins B1, B2, and B3 and moniliformin and for toxicity to 1-day-old ducklings. Most of the members of the A mating population (19 of 20 strains) produced more than 60 micrograms of total fumonisins per g, whereas only 3 of 20 members of the F mating population produced more than trace levels of these toxins and none produced more than 40 micrograms of total fumonisins per g. In addition, only 3 of 20 members of the A mating population produced more than 1 microgram of moniliformin per g (and none produced more than 175 micrograms/g), while all 20 strains of the F mating population produced more than 85 micrograms of this toxin per g and 1 strain produced 10,345 micrograms/g. The duckling toxicity profiles of the strains of the two mating populations were similar, however, and the level of either toxin by itself was not strongly correlated with duckling toxicity. On the basis of our data we think that it is likely that the members of both of these mating populations produce additional toxins that have yet to be chemically identified. These toxins may act singly or synergistically with other compounds to induce the observed duckling toxicity.     

Selection of a strain of Aspergillus for the production of citric acid from pineapple waste in solid-state fermentation

Aspergillus foetidus ACM 3996 (=FRR 3558) and three strains of Aspergillus niger ACM 4992 (=ATCC 9142), ACM 4993 (=ATCC 10577), ACM 4994 (=ATCC 12846) were compared for the production of citric acid from pineapple peel in solid-state fermentation. A. niger ACM 4992 produced the highest amount of citric acid, with a yield of 19.4g of citric acid per 100g of dry fermented pineapple waste under optimum conditions, representing a yield of 0.74g citric acid/g sugar consumed. Optimal conditions were 65% (w/w) initial moisture content, 3% (v/w) methanol, 30°C, an unadjusted initial pH of 3.4, a particle size of 2mm and 5ppm Fe²⁺. Citric acid production was best in flasks, with lower yields being obtained in tray and rotating drum bioreactors.     

Heritability of fumonisin B1 production in Gibberella fujikuroi mating population A.

Fumonisins are mycotoxins produced by strains belonging to several different mating populations of Gibberella fujikuroi (anamorphs, Fusarium section Liseola), a major pathogen of maize and sorghum worldwide. We studied the heritability of fumonisin production in mating population A by crossing fumonisin-producing strains collected from maize and sorghum in the United States with fumonisin-nonproducing strains collected from maize in Nepal. Random ascospore and tetrad progeny from three of these crosses were analyzed by gas chromatography-mass spectrometry and high-performance liquid chromatography for their ability to produce fumonisins on autoclaved cracked maize. In all three crosses, the ability to produce fumonisins, predominately fumonisin B1, segregated as a single gene or group of closely linked genes. Intercrosses between appropriate progeny and parents were poorly fertile, so we could not determine if the apparent single genes that were segregating in each of these crosses were allelic with one another. Mating type and spore-killer traits were scored in some crosses, and each segregated, as expected, as a single gene that was unlinked to the ability to produce fumonisins. We conclude that G. fujikuroi mating population A provides a powerful genetic system for the study of this important fungal toxin.