Aspergillus section Flavi and aflatoxins in Brazilian cassava (Manihot esculenta Crantz) and products.

Aflatoxins are carcinogenic compounds produced by some species of Aspergillus, especially those belonging to Aspergillus section Flavi. Their occurrence in food may start in the field, in the post-harvest, or during storage due to inadequate handling and storage. Because cassava is a staple food for a high percentage of the Brazilian population, we evaluated the presence of aflatoxin-producing species in cassava tubers, cassava products (cassava flour, cassava starch, sour starch, and tapioca flour), and in soil samples collected from cassava fields. In addition, the levels of aflatoxin contamination in cassava products were quantified. A total of 101 samples were analyzed, and 45 strains of Aspergillus section Flavi were isolated. Among the identified species, Aspergillus flavus, Aspergillus arachidicola, Aspergillus novoparasiticus, and Aspergillus parasiticus were found. The majority of strains (73.3%) tested for their aflatoxin-producing ability in synthetic media was positive. Despite that, cassava and cassava products were essentially free of aflatoxins, and only one sample of cassava flour contained traces of AFB1 (0.35 µg/kg).

Fungal Planet description sheets: 1042-1111.

Novel species of fungi described in this study include those from various countries as follows: Antarctica, Cladosporium arenosum from marine sediment sand. Argentina, Kosmimatamyces alatophylus (incl. Kosmimatamyces gen. nov.) from soil. Australia, Aspergillus banksianus, Aspergillus kumbius, Aspergillus luteorubrus, Aspergillus malvicolor and Aspergillus nanangensis from soil, Erysiphe medicaginis from leaves of Medicago polymorpha, Hymenotorrendiella communis on leaf litter of Eucalyptus bicostata, Lactifluus albopicri and Lactifluus austropiperatus on soil, Macalpinomyces collinsiae on Eriachne benthamii, Marasmius vagus on soil, Microdochium dawsoniorum from leaves of Sporobolus natalensis, Neopestalotiopsis nebuloides from leaves of Sporobolus elongatus, Pestalotiopsis etonensis from leaves of Sporobolus jacquemontii, Phytophthora personensis from soil associated with dying Grevillea mccutcheonii. Brazil, Aspergillus oxumiae from soil, Calvatia baixaverdensis on soil, Geastrum calycicoriaceum on leaf litter, Greeneria kielmeyerae on leaf spots of Kielmeyera coriacea. Chile, Phytophthora aysenensis on collar rot and stem of Aristotelia chilensis. Croatia, Mollisia gibbospora on fallen branch of Fagus sylvatica. Czech Republic, Neosetophoma hnaniceana from Buxus sempervirens. Ecuador, Exophiala frigidotolerans from soil. Estonia, Elaphomyces bucholtzii in soil. France, Venturia paralias from leaves of Euphorbia paralias. India, Cortinarius balteatoindicus and Cortinarius ulkhagarhiensis on leaf litter. Indonesia, Hymenotorrendiella indonesiana on Eucalyptus urophylla leaf litter. Italy, Penicillium taurinense from indoor chestnut mill. Malaysia, Hemileucoglossum kelabitense on soil, Satchmopsis pini on dead needles of Pinus tecunumanii. Poland, Lecanicillium praecognitum on insects' frass. Portugal, Neodevriesia aestuarina from saline water. Republic of Korea, Gongronella namwonensis from freshwater. Russia, Candida pellucida from Exomias pellucidus, Heterocephalacria septentrionalis as endophyte from Cladonia rangiferina, Vishniacozyma phoenicis from dates fruit, Volvariella paludosa from swamp. Slovenia, Mallocybe crassivelata on soil. South Africa, Beltraniella podocarpi, Hamatocanthoscypha podocarpi, Coleophoma podocarpi and Nothoseiridium podocarpi (incl. Nothoseiridium gen. nov.) from leaves of Podocarpus latifolius, Gyrothrix encephalarti from leaves of Encephalartos sp., Paraphyton cutaneum from skin of human patient, Phacidiella alsophilae from leaves of Alsophila capensis, and Satchmopsis metrosideri on leaf litter of Metrosideros excelsa. Spain, Cladophialophora cabanerensis from soil, Cortinarius paezii on soil, Cylindrium magnoliae from leaves of Magnolia grandiflora, Trichophoma cylindrospora (incl. Trichophoma gen. nov.) from plant debris, Tuber alcaracense in calcareus soil, Tuber buendiae in calcareus soil. Thailand, Annulohypoxylon spougei on corticated wood, Poaceascoma filiforme from leaves of unknown Poaceae. UK, Dendrostoma luteum on branch lesions of Castanea sativa, Ypsilina buttingtonensis from heartwood of Quercus sp. Ukraine, Myrmecridium phragmiticola from leaves of Phragmites australis. USA, Absidia pararepens from air, Juncomyces californiensis (incl. Juncomyces gen. nov.) from leaves of Juncus effusus, Montagnula cylindrospora from a human skin sample, Muriphila oklahomaensis (incl. Muriphila gen. nov.) on outside wall of alcohol distillery, Neofabraea eucalyptorum from leaves of Eucalyptus macrandra, Diabolocovidia claustri (incl. Diabolocovidia gen. nov.) from leaves of Serenoa repens, Paecilomyces penicilliformis from air, Pseudopezicula betulae from leaves of leaf spots of Populus tremuloides. Vietnam, Diaporthe durionigena on branches of Durio zibethinus and Roridomyces pseudoirritans on rotten wood. Morphological and culture characteristics are supported by DNA barcodes.

A taxonomic and phylogenetic revision of Penicillium section Aspergilloides.

Species belonging to Penicillium section Aspergilloides have a world-wide distribution with P. glabrum, P. spinulosum and P. thomii the most well-known species of this section. These species occur commonly and can be isolated from many substrates including soil, food, bark and indoor environments. The taxonomy of these species has been investigated several times using various techniques, but species delimitation remains difficult. In the present study, 349 strains belonging to section Aspergilloides were subjected to multilocus molecular phylogenetic analyses using partial ß-tubulin (BenA), calmodulin (CaM) and RNA polymerase II second largest subunit (RPB2) sequences. Section Aspergilloides is subdivided into 12 clades and 51 species. Twenty-five species are described here as new and P. yezoense, a species originally described without a Latin diagnosis, is validated. Species belonging to section Aspergilloides are phenotypically similar and most have monoverticillate conidiophores and grow moderately or quickly on agar media. The most important characters to distinguish these species were colony sizes on agar media, growth at 30 °C, ornamentation and shape of conidia, sclerotium production and stipe roughness.

An extension of the Coconut Cream Agar method to screen Penicillium citrinum isolates for citrinin production.

A simple and rapid screening method was developed for the detection of citrinin in fungal cultures using Coconut Cream Agar (CCA) described previously for detecting aflatoxin and ochratoxin A. Fifteen isolates of Penicillium citrinum were inoculated onto CCA and incubated at 25 and 30°C for 10 days. All isolates produced a distinct yellow green fluorescence on CCA when the reverse side of the agar plates were viewed under long wavelength UV light. Detection was optimal at 25°C after four to 5 days of incubation. Isolates positive by the CCA method also tested positive for citrinin production by the TLC agar plug method after growth on CCA, Czapek yeast extract agar and yeast extract sucrose agar. Control cultures were negative by both methods, indicating that the CCA Petri dish method was suitable for screening cultures for citrinin production.

Growth and mycotoxin production by fungi in atmospheres containing 80% carbon dioxide and 20% oxygen.

The effect of atmosphere containing 80% CO(2) and 20% O(2) on growth of Mucor plumbeus, Fusarium oxysporum, Byssochlamys fulva, Byssochlamys nivea, Penicillium commune, Penicillium roqueforti, Aspergillus flavus, Eurotium chevalieri and Xeromyces bisporus was investigated. Production of aflatoxin by A. flavus, patulin by B. nivea, roquefortine C by P. roqueforti, and cyclopiazonic acid by P. commune was also studied. Fungal growth was evaluated by three methods: colony diameter, hyphal length or mycelium dry weight and ergosterol content. Among the nine fungal species examined, two E. chevalieri and X. bisporus, did not grow under these conditions. In this study, fungi differed in their response to modified atmospheres in biomass, ergosterol content, mycotoxin production and morphology. Reductions of 57.8-96.9%, 73.7-99.6% and 91.5-99.9% were obtained in colony diameter, hyphal length and ergosterol content, respectively, under this atmosphere compared to air. Ergosterol content was more affected in most species than other measurements. Patulin, cyclopiazonic acid and roquefortine C were produced in this atmosphere, although levels were very low and aflatoxin was not produced at all. Growth was quite extensive as measured by colony diameters, but hyphal lengths were low and ergosterol production was also affected in all species of this study.

Growth and mycotoxin production by food spoilage fungi under high carbon dioxide and low oxygen atmospheres.

The influence of high carbon dioxide and low oxygen concentrations on growth by the foodborne fungal species, Mucor plumbeus, Fusarium oxysporum, Byssochlamys fulva, Byssochlamys nivea, Penicillium commune, Penicillium roqueforti, Aspergillus flavus, Eurotium chevalieri and Xeromyces bisporus was investigated. Production of aflatoxin by A. flavus, patulin by B. nivea and roquefortine C by P. roqueforti was also studied. Fungal growth was evaluated under atmospheres consisting of 20, 40 and 60% CO(2) plus <0.5% O(2), on two media, Czapek Yeast Extract agar and Potato Dextrose agar. Several methods for measuring fungal growth were used: colony diameter, ergosterol content, hyphal length and/or mycelium dry weight. Among the nine species, three groups were distinguished with respect to their growth responses under modified atmospheres: (i) species which did not grow in 20% CO(2) <0.5% O(2) (P. commune, E. chevalieri and X. bisporus); (ii) species which grew in 20% CO(2) <0.5% O(2), but not 40% CO(2) <0.5% O(2) (P. roqueforti and A. flavus); (iii) species which grew in 20%, 40% and 60% CO(2) <0.5% O(2) (M. plumbeus, F. oxysporum, B. fulva and B. nivea). Facultatively anaerobic behaviour was observed in these last four species, which grew under the same conditions as the obligate anaerobe, Clostridium sporogenes. The production of aflatoxin, patulin, and roquefortine C was greatly reduced under all of the atmospheres tested.

Nomenclatural considerations in naming species of Aspergillus and its teleomorphs.

The nomenclature of Aspergillus is important in many fields of research and therefore the strategies for stable and efficient naming are important. The conservation of species names as accepted by the Aspergillus community is described. Published lists of accepted names provide that people who use Aspergillus and Penicillium taxonomies need no longer fear the overturning of names currently used. Aspergillus is a good example of a genus where the naming of both anamorph and teleomorph has been applied and arguments are given for maintaining the system of dual nomenclature. A protocol for describing new taxa in Aspergillus and their teleomorphs is proposed, including the availability of living ex type cultures, deposit of type cultures in at least two recognised culture collections, deposits of sequence data in specialised data bases and registration of the new names in MycoBank.

Evaluation of certain food additives and contaminants.

This report represents the conclusions of a Joint FAO/WHO Expert Committee convened to evaluate the safety of various food additives, including flavouring agents, with a view to recommending acceptable daily intakes (ADIs) and to preparing specifications for identity and purity. The Committee also evaluated the risk posed by two food contaminants, with the aim of advising on risk management options for the purpose of public health protection. The first part of the report contains a general discussion of the principles governing the toxicological evaluation and assessment of intake of food additives (in particular flavouring agents) and contaminants. A summary follows of the Committee's evaluations of technical, toxicological and intake data for certain food additives (acidified sodium chlorite, asparaginase from Aspergillus oryzae expressed in Aspergillus oryzae, carrageenan and processed Eucheuma seaweed, cyclotetraglucose and cyclotetraglucose syrup, isoamylase from Pseudomonas amyloderamosa, magnesium sulfate, phospholipase A1 from Fusarium venenatum expressed in Aspergillus oryzae, sodium iron(III) ethylenediaminetetraacetic acid (EDTA) and steviol glycosides); eight groups of related flavouring agents (linear and branched-chain aliphatic, unsaturated, unconjugated alcohols, aldehydes, acids and related esters; aliphatic acyclic and alicyclic terpenoid tertiary alcohols and structurally related substances; simple aliphatic and aromatic sulfides and thiols; aliphatic acyclic dials, trials and related substances; aliphatic acetals; sulfur-containing heterocyclic compounds; aliphatic and aromatic amines and amides; and aliphatic alicyclic linear alpha, beta -unsaturated di- and trienals and related alcohols, acids and esters); and two food contaminants (aflatoxin and ochratoxin A). Specifications for the following food additives were revised: maltol and ethyl maltol, nisin preparation, pectins, polyvinyl alcohol, and sucrose esters of fatty acids. Specifications for the following flavouring agents were revised: maltol and ethyl maltol, maltyl isobutyrate, 3-acetyl-2,5-dimethylfuran and 2,4,5-trimethyl-delta-oxazoline (Nos 1482, 1506 and 1559), and monomenthyl glutarate (No. 1414), as well as the method of assay for the sodium salts of certain flavouring agents. Annexed to the report are tables summarizing the Committee's recommendations for intakes and toxicological evaluations of the food additives and contaminants considered.

Mycotoxins in Australia: biocontrol of aflatoxin in peanuts.

The major mycotoxin problem in Australia is the formation of aflatoxins in peanuts by Aspergillus flavus and A. parasiticus. This is controlled by good farm management practice, segregation into grades on aflatoxin content at intake to shelling facilities, colour sorting and aflatoxin assays. A second problem is the potential presence of ochratoxin A in grapes and grape products, resulting from infection by Aspergillus carbonarius. Good quality control before and during wine making ensures ochratoxin A is kept to very low levels, but in dried vine fruit, ochratoxin A levels may be higher. Biocontrol by competitive exclusion has been developed as the most promising means of controlling aflatoxins in peanuts. Some details of the process are given, including some basic laboratory experiments.

Australian research on ochratoxigenic fungi and ochratoxin A.

The presence of the mycotoxin, ochratoxin A (OTA), has been reported in Australian grape products. Comprehensive surveys of Australian wines have determined that the frequency and level of OTA contamination are low. Aspergillus carbonarius is the primary OTA-producing species associated with grapes in Australia, and all isolates tested to date produce OTA. Aspergillus niger is isolated more frequently from vineyards, however, few strains produce OTA. A. carbonarius and A. niger exist as saprophytes in the top layer of soil beneath vines, from where they are thought to be blown onto bunches. The level of A. carbonarius in soil may be reduced by temperatures above or below the optimum temperature for survival (25 degrees C), by high soil moisture content, and by modifications to tillage and mulching practices. A. carbonarius is an opportunistic pathogen of damaged berries. In the absence of damage, spores may exist on berry surfaces without causing visible rots. Aspergillus rots are associated with black Aspergillus species, primarily A. niger, A. carbonarius and A. aculeatus. The potential for such rots is increased with berry damage, inoculum coverage and berry maturity. Susceptibility to berry splitting is related, in part, to bunch structure, and may be variety-dependent or influenced by rainfall, irrigation and canopy management. Black Aspergillus spp. are closely associated with berries near the main stem of the bunch. During winemaking, around 80% of the OTA initially present in grapes is removed, primarily with the skins and pulp during pressing. Additional reductions occur with the removal of precipitated grape and yeast solids. Bentonite in white wine and yeast hulls in red wine were the most effective non-carbonaceous fining agents for the removal of OTA.

The source of ochratoxin A in Brazilian coffee and its formation in relation to processing methods.

A total of 408 Brazilian coffee samples was examined during the 1999 and 2000 coffee harvest seasons for the presence of ochratoxin A (OA) and fungi with the potential to produce it. Samples came from four regions: Alta Paulista (western area of São Paulo State), Sorocabana (southwest São Paulo State), Alta Mogiana (northeast São Paulo State) and Cerrado Mineiro (western area of Minas Gerais State). Cherries and beans were examined at different stages: immature, mature and overripe cherries from trees, overripe cherries from the ground and beans during drying and storage on the farm. For mycological studies, the cherries and beans were surface disinfected with chlorine, plated on Dichloran 18% Glycerol Agar at 25 degrees C for 5-7 days and analysed for the presence of Aspergillus ochraceus and closely related species, A. carbonarius and A. niger. More than 800 isolates of fungi belonging to these species were identified and studied for the ability to produce OA using the agar plug technique and thin layer chromatography (TLC). A. niger was the species found most commonly (63% of isolates of these three species), but only 3% of them produced OA. A. ochraceus also occurred commonly (31% of isolates), and 75% of those studied were capable of OA production, a much higher percentage than reported elsewhere. A. carbonarius was found (6% of isolates) only in Alta Paulista, the hottest region studied, and only from beans in the drying yard or in storage. However, 77% of the A. carbonarius isolates were capable of producing OA. Average infection rates for cherries taken from trees were very low, but were higher in fruit taken from the ground, from the drying yard and from storage, indicating infection by toxigenic species after harvest. The average OA content in 135 samples of mature cherries from trees, overripe from trees, overripe from the ground, drying yard and storage was 0.1, <0.2, 1.6, 2.1 and 3.3 microg/kg, respectively. Although individual OA levels varied widely, only 9 of the 135 samples analysed exceeded 5 microg/kg OA, with one sample of poor quality dried coffee in excess of 100 microg/kg OA. The causes of high contamination were investigated on the farms concerned and several critical points were found, relating both to local climatic conditions and the drying processes used.

Growth of fungi and mycotoxin production on cheese under modified atmospheres.

The use of modified atmospheres to prevent fungal growth and mycotoxin production in cheese was evaluated. Eight fungal species: Mucor plumbeus, Fusarium oxysporum, Byssochlamys fulva, B. nivea, Penicillium commune, P. roqueforti, Aspergillus flatus and Eurotium chevalieri were inoculated onto cheese and incubated under conditions of decreasing concentrations of O2 (5% to < 0.5%) and increasing concentrations of CO2 (20-40%). Fungal growth was measured by colony diameter and ergosterol content. All fungi examined grew in atmospheres containing 20% and 40% CO2 with 1% or 5% O2, but growth was reduced by 20-80%, depending on species, compared with growth in air. The formation of aflatoxins B1 and B2, roquerfortine C and cyclopiazonic acid was greatly decreased but not totally inhibited in these atmospheres. At 20% or 40% CO2 with < 0.5% O2, only B. nivea exhibited growth, which was very slow. Growth of F. oxysporum, B. fulca, P. commune and A. flavus showed good correlations between colony diameter and ergosterol content. However, for the other species correlations were inconsistent.

Systemic mycosis due to Aspergillus deflectus in a dog.

A 4-year-old, entire female, German Shepherd Dog was referred with a 3-month history of right foreleg lameness that partially responded to nonsteroidal anti-inflammatory and antimicrobial therapy. The bitch lost weight, was polydipsic and had reduced exercise tolerance. On referral, the animal was in poor condition, pyrexic and exhibited moderate pain on full extension of the right shoulder. Blood, urine and joint fluid were obtained and radiographs were taken of the right shoulder and chest. The bitch was lymphopaenic, hyperfibrinogenaemic, hyperglobulinaemic, mildly azotaemic, mildly proteinuric and isosthenuric. Branching fungal hyphae were present in the urine. On radiography, the thorax contained a large ventral mediastinal mass and the humeral head had extensive areas of radiolucency. An aspirate from the right humeroscapular joint exhibited branched fungal hyphae and numerous neutrophils and macrophages. A diagnosis of disseminated mycosis was made and euthanasia was performed. At necropsy, numerous caseating granulomas were present, especially in the kidneys, adrenal glands, heart and lymph nodes. Extensive osteomyelitis involved the head of the right humerus, the sternebrae and the fifth intervertebral disc. Fungal hyphae were detected in sections of granulomas in all affected organs and a diagnosis of disseminated fungal granulomatosis was made. Aspergillus deflectus was readily isolated from affected lymph nodes, but confirming its identity as A deflectus using standard procedures proved difficult. The identity of the fungus was finally confirmed by sequencing part of the 185 rRNA of the isolate. This is the first report in Australia of a disseminated mycosis caused by A deflectus. Previously, the involvement of A deflectus as a cause of disseminated mycosis was limited to 5 cases from the West Coast of the USA, four of which occurred in German Shepherd Dogs.

Toxigenic fungi and mycotoxins.

Growth of commonly occurring filamentous fungi in foods may result in production of toxins known as mycotoxins, which can cause a variety of ill effects in humans, from allergic responses to immunosuppression and cancer. The most important mycotoxins are aflatoxins, ochratoxin A, fumonisins, trichothecenes and zearalenone. Aflatoxins are potent carcinogens and, in association with hepatitis B virus, are responsible for many thousands of human deaths per annum, mostly in non-industrialised tropical countries. Ochratoxin A is a probable carcinogen, and may cause urinary tract cancer and kidney damage in people from northern and eastern Europe. Fumonisins appear to be the cause of oesophageal cancer in southern Africa, parts of China and elsewhere. Trichothecenes are highly immunosuppressive and zearalenone causes oestrogenic effects in animals and man. Currently available records and statistics do not reflect the major role played by mycotoxins in mortality attributable to food-borne micro-organisms.

Toxigenic fungi: which are important?

Growth of commonly occurring filamentous fungi in foods may result in production of mycotoxins, which can cause a variety of ill effects in humans, from allergic responses to immunosuppression and cancer. According to experts, five kinds of mycotoxins are important in human health around the world: aflatoxins, ochratoxin A, fumonisins, certain trichothecenes, and zearalenone. These toxins are produced by only a few species of fungi, in a limited range of commodities. Aflatoxins are potent carcinogens, produced by Aspergillus flavus and A. parasiticus in peanuts, maize and some other nuts and oilseeds. Ochratoxin A is a kidney toxin and probable carcinogen. It is produced by Penicillium verrucosum in cereal grains in cold climates, by A. carbonarius in grapes, wines and vine fruits, and by A. ochraceus sometimes in coffee beans. Fumonisins, which may cause oesophageal cancer, are formed by Fusarium moniliforme and F. proliferatum, but only in maize. Trichothecenes are highly immunosuppressive and zearalenone causes oestrogenic effects; both are produced by F. graminearum and related species. Current reporting probably underestimates the effect of mycotoxins as a cause of human mortality.

Mycotoxins and toxigenic fungi.

Mycotoxins and the fungi that produce them are of increasing importance as causes of human illness, but the diseases produced remain poorly understood at the clinical level. This paper explores four aspects: the increase of interest in ochratoxin A, factors affecting mycotoxin production, toxicology of the major mycotoxins, and the identification of Penicillium species which cause food spoilage and are important in indoor air.

Cryptic speciation and recombination in the aflatoxin-producing fungus Aspergillus flavus.

Aspergillus flavus, like approximately one-third of ascomycete fungi, is thought to be cosmopolitan and clonal because it has uniform asexual morphology. A. flavus produces aflatoxin on nuts, grains, and cotton, and assumptions about its life history are being used to develop strategies for its biological control. We tested the assumptions of clonality and conspecificity in a sample of 31 Australian isolates by assaying restriction site polymorphisms from 11 protein encoding genes and DNA sequences from five of those genes. A. flavus isolates fell into two reproductively isolated clades (groups I and II). The lack of concordance among gene genealogies among isolates in one of the clades (group I) was consistent with a history of recombination. Our analysis included five strains of the closely related industrial fungus A. oryzae, all of which proved to be clonally related to group I.