Deep Genotypic Species Delimitation of Aspergillus Section Flavi Isolated from Brazilian Foodstuffs and the Description of Aspergillus annui sp. nov. and Aspergillus saccharicola sp. nov.

Aspergillus section Flavi is a fungal group that is important in food because it contains spoilage and potentially aflatoxigenic species. Aflatoxins are metabolites that are harmful to human and animal health and have been recognized as the primary natural contaminant in food. Therefore, recognizing the biodiversity of this group in food is necessary to reduce risks to public health. Our study aimed to investigate the diversity of Aspergillus section Flavi isolated from Brazilian foodstuffs such as cassava, sugarcane, black pepper, paprika, Brazil nuts, yerba-mate, peanuts, rice, and corn. A polyphasic approach integrating phenotypic data and multilocus genotypic analyses (CaM, BenA, and RPB2) was performed for 396 strains. Two new species in the Aspergillus subgenus Circumdati section Flavi are proposed using maximum-likelihood analysis, Bayesian inference, and coalescence-based methods: Aspergillus saccharicola sp. nov. and Aspergillus annui sp. nov. A. saccharicola sp. nov. belongs to the series Flavi, is a potentially aflatoxigenic species (B1, B2, G1, and G2), closely related to Aspergillus arachidicola, and was found mostly in sugarcane. A. annui sp. nov. was isolated from samples of sweet paprika. To accommodate A. annui sp. nov., a new series Annuorum was proposed.

Diversity within Aspergillus niger Clade and Description of a New Species: Aspergillus vinaceus sp. nov.

Diversity of species within Aspergillus niger clade, currently represented by A. niger sensu stricto and A. welwitshiae, was investigated combining three-locus gene sequences, Random Amplified Polymorphic DNA, secondary metabolites profile and morphology. Firstly, approximately 700 accessions belonging to this clade were investigated using calmodulin gene sequences. Based on these sequences, eight haplotypes were clearly identified as A. niger (n = 247) and 17 as A. welwitschiae (n = 403). However, calmodulin sequences did not provide definitive species identities for six haplotypes. To elucidate the taxonomic position of these haplotypes, two other loci, part of the beta-tubulin gene and part of the RNA polymerase II gene, were sequenced and used to perform an analysis of Genealogical Concordance Phylogenetic Species Recognition. This analysis enabled the recognition of two new phylogenetic species. One of the new phylogenetic species showed morphological and chemical distinguishable features in comparison to the known species A. welwitschiae and A. niger. This species is illustrated and described as Aspergillus vinaceus sp. nov. In contrast to A. niger and A. welwitschiae, A. vinaceus strains produced asperazine, but none of them were found to produce ochratoxin A and/or fumonisins. Sclerotium production on laboratory media, which does not occur in strains of A. niger and A. welwitschiae, and strictly sclerotium-associated secondary metabolites (14-Epi-hydroxy-10,23-dihydro-24,25-dehydroaflavinine; 10,23-Dihydro-24,25-dehydroaflavinine; 10,23-Dihydro-24,25-dehydro-21-oxo-aflavinine) were found in A. vinaceus. The strain type of A. vinaceus sp. nov. is ITAL 47,456 (T) (=IBT 35556).

Fungal communities in rice cultivated in different Brazilian agroclimatic zones: From field to market.

Rice is one of the most consumed cereals in Brazil and around the world. Due to the major health impact of rice consumption on populations, studies about its quality have great importance. The present study determined the mycobiota of soil, field, processing and market rice samples from two production systems in Brazil, dryland in the state of Maranhão and wetland in the state of Rio Grande do Sul. These areas are distinct agroclimatic zones. A total of 171 rice and 23 soil samples were analyzed. A high differentiation was observed in the composition of the fungal communities found in the two production systems, as the wetland presented greater fungal incidence and biodiversity. It was observed that toxigenic species from Aspergillus section Flavi and Fusarium, present in the field, may infect rice grains pre or postharvest and may persist into the final product.

Occurrence of Aspergillus section Flavi and aflatoxins in Brazilian rice: From field to market.

The guarantee of the high quality of rice is of utmost importance because any toxic contaminant may affect consumer health, especially in countries such as Brazil where rice is part of the daily diet. A total of 187 rice samples, from field, processing and market from two different production systems, wetland from the state of Rio Grande do Sul, dryland, from the state of Maranhão and market samples from the state of São Paulo, were analyzed for fungi belonging to Aspergillus section Flavi and the presence of aflatoxins. Twenty-three soil samples from wetland and dryland were also analyzed. A total of 383 Aspergillus section Flavi strains were isolated from rice and soil samples. Using a polyphasic approach, with phenotypic (morphology and extrolite profiles) and molecular data (beta-tubulin gene sequences), five species were identified: A. flavus, A. caelatus, A. novoparasiticus, A. arachidicola and A. pseudocaelatus. This is the first report of these last three species from rice and rice plantation soil. Only seven (17%) of the A. flavus isolates produced type B aflatoxins, but 95% produced kojic acid and 69% cyclopiazonic acid. Less than 14% of the rice samples were contaminated with aflatoxins, but two of the market samples were well above the maximum tolerable limit (5µg/kg), established by the Brazilian National Health Surveillance Agency.

Aspergillus labruscus sp. nov., a new species of Aspergillus section Nigri discovered in Brazil.

A novel fungal species, Aspergillus labruscus sp. nov., has been found in Brazil during an investigation of the fungal species present on the surface of grape berries (Vitis labrusca L.) for use in the production of concentrated grape juice. It seems to be associated to V. labrusca, and has never been recovered from Vitis vinifera. This new species belonging to Aspergillus subgenus Circumdati section Nigri is described here using morphological characters, extrolite profiling, partial sequence data from the BenA and CaM genes, and internal transcribed spacer sequences of ribosomal DNA. Phenotypic and molecular data enabled this novel species to be clearly distinguished from other black aspergilli. A. labruscus sp. nov. is uniseriate, has yellow mycelium, poor sporulation on CYA at 25 °C, abundant salmon to pink sclerotia and rough conidia. Neoxaline and secalonic acid D were consistently produced by isolates in this taxon. The type strain of A. labruscus sp. nov. is CCT 7800 (T) = ITAL 22.223 (T) = IBT 33586 (T).

Biodiversity of mycobiota throughout the Brazil nut supply chain: From rainforest to consumer.

A total of 172 Brazil nut samples (114 in shell and 58 shelled) from the Amazon rainforest region and São Paulo state, Brazil was collected at different stages of the Brazil nut production chain: rainforest, street markets, processing plants and supermarkets. The mycobiota of the Brazil nut samples were evaluated and also compared in relation to water activity. A huge diversity of Aspergillus and Penicillium species were found, besides Eurotium spp., Zygomycetes and dematiaceous fungi. A polyphasic approach using morphological and physiological characteristics, as well as molecular and extrolite profiles, were studied to distinguish species among the more important toxigenic ones in Aspergillus section Flavi and A. section Nigri. Several metabolites and toxins were found in these two sections. Ochratoxin A (OTA) was found in 3% of A. niger and 100% of A. carbonarius. Production of aflatoxins B and G were found in all isolates of A. arachidicola, A. bombycis, A. nomius, A. pseudocaelatus and A. pseudonomius, while aflatoxin B was found in 38% of A. flavus and all isolates of A. pseudotamarii. Cyclopiazonic acid (CPA) was found in A. bertholletius (94%), A. tamarii (100%), A. caelatus (54%) and A. flavus (41%). Tenuazonic acid, a toxin commonly found in Alternaria species was produced by A. bertholletius (47%), A. caelatus (77%), A. nomius (55%), A. pseudonomius (75%), A. arachidicola (50%) and A. bombycis (100%). This work shows the changes of Brazil nut mycobiota and the potential of mycotoxin production from rainforest to consumer, considering the different environments which exist until the nuts are consumed.

Occurrence and fumonisin B2 producing potential of Aspergillus section Nigri in Brazil nuts.

Bertholletia excelsa is the tree that produces Brazil nuts which have vast economic importance in the Amazon region and as an export commodity. The aim of this study was to assess the presence of Aspergillus section Nigri in Brazil nut samples at different stages of its production chain and to verify the toxigenic potential for fumonisin B2 (FB2) production of these isolates along with the presence of this mycotoxin in Brazil nut samples. The fungal infection ranged from 0 to 80% at the different stages of the harvest and processing chain and the water activity of the nuts from 0.273 to 0.994. A total of 1052 A. section Nigri strains were isolated from Brazil nuts and 200 strains were tested for their ability to produce FB2: 41 strains (20.5%) were FB2 producers with concentrations ranging from 0.09 to 37.25 mg/kg; 2 strains (1%) showed traces of FB2, less than the detection limit (0.08 mg/kg); and 157 (78.5%) were not FB2 producers. Although several samples showed high contamination by A. section Nigri, no sample was contaminated by FB2.

Penicillium excelsum sp. nov from the Brazil Nut Tree Ecosystem in the Amazon Basin'.

A new Penicillium species, P. excelsum, is described here using morphological characters, extrolite and partial sequence data from the ITS, ß-tubulin and calmodulin genes. It was isolated repeatedly using samples of nut shells and flowers from the brazil nut tree, Bertolletia excelsa, as well as bees and ants from the tree ecosystem in the Amazon rainforest. The species produces andrastin A, curvulic acid, penicillic acid and xanthoepocin, and has unique partial ß-tubulin and calmodulin gene sequences. The holotype of P. excelsum is CCT 7772, while ITAL 7572 and IBT 31516 are cultures derived from the holotype.

Brazil nuts are subject to infection with B and G aflatoxin-producing fungus, Aspergillus pseudonomius.

The exploitation of the Brazil nut is one of the most important activities of the extractive communities of the Amazon rainforest. However, its commercialization can be affected by the presence of aflatoxins produced by fungi, namely Aspergillus section Flavi. In the present study, we investigated a collection of Aspergillus nomius strains isolated from Brazil nuts using different approaches, including morphological characters, RAPD and AFLP profiles, partial ß-tubulin and calmodulin nucleotide sequences, aflatoxin patterns, as well as tolerance to low water activity in cultured media. Results showed that most of the isolates do belong to A. nomius species, but a few were re-identified as Aspergillus pseudonomius, a very recently described species. The results of the analyses of molecular variance, as well as the high pairwise FST values between A. nomius and A. pseudonomius suggested the isolation between these two species and the inexistence of gene flow. Fixed interspecific nucleotide polymorphisms at ß-tubulin and calmodulin loci are presented. All A. pseudonomius strains analyzed produced aflatoxins AFB1, AFB2, AFG1 and AFG2. This study contains the first-ever report on the occurrence in Brazil nuts of A. pseudonomius. The G-type aflatoxins and the mycotoxin tenuazonic acid are reported here for the first time in A. pseudonomius.

The biodiversity of Aspergillus section Flavi in brazil nuts: from rainforest to consumer.

A total of 288 brazil nut samples (173 kernel and 115 shell) from the Amazon rainforest region and São Paulo State, Brazil were collected at different stages of brazil nut production. Samples were analysed for: percentages of aflatoxigenic fungal species and potential for aflatoxin production and presence of aflatoxins. Aspergillus nomius was the most common species found (1235 isolates) which amounted to 30% of the total species with potential to produce aflatoxins. This species is of concern since 100% of all isolates produced aflatoxins B(1), B(2), G(1) and G(2). Aspergillus flavus was almost equally common (1212 isolates) although only 46% produced aflatoxins under laboratory conditions, and only aflatoxins B(1) and B(2). Low number of other species with the potential to produce aflatoxins was isolated: Aspergillus arachidicola and Aspergillus bombycis produced B and G aflatoxins whilst Aspergillus pseudotamarii produced only aflatoxin B(1). The total aflatoxin levels found in samples taken from the rainforests was 0.7 µg/kg, from processing plants before and after sorting 8.0 and 0.1 µg/kg respectively, from street markets in the Amazon region 6.3 µg/kg and from supermarkets in São Paulo State 0.2 µg/kg. Processing, which included manual or mechanical sorting and drying at 60°C for 30 to 36 h, eliminated on average more than 98% of total aflatoxins. These results showed that sorting is a very effective way to decrease aflatoxin content in brazil nuts.

Aspergillus bertholletius sp. nov. from Brazil nuts.

During a study on the mycobiota of brazil nuts (Bertholletia excelsa) in Brazil, a new Aspergillus species, A. bertholletius, was found, and is described here. A polyphasic approach was applied using morphological characters, extrolite data as well as partial ß-tubulin, calmodulin and ITS sequences to characterize this taxon. A. bertholletius is represented by nineteen isolates from samples of brazil nuts at various stages of production and soil close to Bertholletia excelsa trees. The following extrolites were produced by this species: aflavinin, cyclopiazonic acid, kojic acid, tenuazonic acid and ustilaginoidin C. Phylogenetic analysis using partial ß-tubulin and camodulin gene sequences showed that A. bertholletius represents a new phylogenetic clade in Aspergillus section Flavi. The type strain of A. bertholletius is CCT 7615 ( = ITAL 270/06 = IBT 29228).

The effect of cocoa fermentation and weak organic acids on growth and ochratoxin A production by Aspergillus species.

The acidic characteristics of cocoa beans have influence on flavor development in chocolate. Cocoa cotyledons are not naturally acidic, the acidity comes from organic acids produced by the fermentative microorganisms which grow during the processing of cocoa. Different concentrations of these metabolites can be produced according to the fermentation practices adopted in the farms, which could affect the growth and ochratoxin A production by fungi. This work presents two independent experiments carried out to investigate the effect of some fermentation practices on ochratoxin A production by Aspergillus carbonarius in cocoa, and the effect of weak organic acids such as acetic, lactic and citric at different pH values on growth and ochratoxin A production by A. carbonarius and Aspergillus niger in culture media. A statistical difference (ρ<0.05) in the ochratoxin A level in the cured cocoa beans was observed in some fermentation practices adopted. The laboratorial studies demonstrate the influence of organic acids on fungal growth and ochratoxin A production, with differences according to the media pH and the organic acid present. Acetic acid was the most inhibitory acid against A. carbonarius and A. niger. From the point of view of food safety, considering the amount of ochratoxin A produced, fermentation practices should be conducted towards the enhancement of acetic acid, although lactic and citric acids also have an important role in lowering the pH to improve the toxicity of acetic acid.

Mycobiota of cocoa: from farm to chocolate.

The present work was carried out to study the mycobiota of cocoa beans from farm to chocolate. Four hundred and ninety-four samples were analyzed at various stages of cocoa processing: (i) primary stage at the farm (fermentation, drying, and storage), (ii) secondary stage at processing (testa, nibs, liquor, butter, cake and powder) and (iii) the final chocolate product (dark, milk, white and powdered) collected from retail outlets. Direct plating or dilution plating on Dichloran 18% Glycerol agar were used for cocoa beans and processed product analyses, respectively. Fungi were isolated and identified using different keys of identification. The largest numbers and diversity of fungi were observed in the samples collected at the farm, especially during drying and storage. The species with the highest occurrence among samples were: Absidia corymbifera, Aspergillus sp. nov., A. flavus, Penicillium paneum and yeasts. A total of 1132 potentially toxigenic fungi were isolated from the following species or species groups: A. flavus, Aspergillus parasiticus, Aspergillus nomius, Aspergillus niger group, Aspergillus carbonarius and Aspergillus ochraceus group. The highest percentage of toxigenic fungi was found at the drying and storage stages. The industrial processing reduced the fungal contamination in all fractions and no fungi were found in the final chocolate products. The knowledge of which fungi are dominant at each processing stage of cocoa provides important data about their ecology. This understanding leads to a reduction in fungal spoilage and mycotoxin production in this product.

Penicillium araracuarense sp. nov., Penicillium elleniae sp. nov., Penicillium penarojense sp. nov., Penicillium vanderhammenii sp. nov. and Penicillium wotroi sp. nov., isolated from leaf litter.

Several species of the genus Penicillium were isolated during a survey of the mycobiota of leaf litter and soil in Colombian Amazon forest. Five species, Penicillium penarojense sp. nov. (type strain CBS 113178(T) = IBT 23262(T)), Penicillium wotroi sp. nov. (type strain CBS 118171(T) = IBT 23253(T)), Penicillium araracuarense sp. nov. (type strain CBS 113149(T) = IBT 23247(T)), Penicillium elleniae sp. nov. (type strain CBS 118135(T) = IBT 23229(T)) and Penicillium vanderhammenii sp. nov. (type strain CBS 126216(T) = IBT 23203(T)) are described here as novel species. Their taxonomic novelty was determined using a polyphasic approach, combining phenotypic, molecular (ITS and partial ß-tubulin sequences) and extrolite data. Phylogenetic analyses showed that each novel species formed a unique clade for both loci analysed and that they were most closely related to Penicillium simplicissimum, Penicillium janthinellum, Penicillium daleae and Penicillium brasilianum. An overview of the phylogeny of this taxonomically difficult group is presented, and 33 species are accepted. Each of the five novel species had a unique extrolite profile of known and uncharacterized metabolites and various compounds, such as penicillic acid, andrastin A, pulvilloric acid, paxillin, paspaline and janthitrem, were commonly produced by these phylogenetically related species. The novel species had a high growth rate on agar media, but could be distinguished from each other by several macro- and microscopical characteristics.

Two novel aflatoxin-producing Aspergillus species from Argentinean peanuts.

Two novel species from Aspergillus section Flavi from different species of Arachis (peanuts) in Argentina are described as Aspergillus arachidicola sp. nov. and Aspergillus minisclerotigenes sp. nov. Their novel taxonomic status was determined using a polyphasic taxonomic approach with phenotypic (morphology and extrolite profiles) and molecular (beta-tubulin and calmodulin gene sequences) characters. A. minisclerotigenes resembles Aspergillus flavus and Aspergillus parvisclerotigenus in producing aflatoxins B(1) and B(2), cyclopiazonic acid, kojic acid and aspergillic acid, but in addition it produces aflatoxins G(1) and G(2), aflavarins, aflatrem, aflavinines, parasiticolides and paspaline. This species also includes several isolates previously assigned to A. flavus group II and three Australian soil isolates. A. arachidicola produces aflatoxins B(1), B(2), G(1) and G(2), kojic acid, chrysogine and parasiticolide, and some strains produce aspergillic acid. The type strain of A. arachidicola is CBS 117610(T) =IBT 25020(T) and that of A. minisclerotigenes is CBS 117635(T) =IBT 27196(T). The Mycobank accession numbers for Aspergillus minisclerotigenes sp. nov. and Aspergillus arachidicola sp. nov. are respectively MB 505188 and MB 505189 (http://www.mycobank.org).

Aspergillus brasiliensis sp. nov., a biseriate black Aspergillus species with world-wide distribution.

A novel species, Aspergillus brasiliensis sp. nov., is described within Aspergillus section Nigri. This species can be distinguished from other black aspergilli based on intergenic transcribed region, beta-tubulin and calmodulin gene sequences, by amplified fragment length polymorphism analysis and by extrolite profiles. A. brasiliensis isolates produced naphtho-gamma-pyrones, tensidol A and B and pyrophen in common with Aspergillus niger and Aspergillus tubingensis, but also several unique compounds, justifying their treatment as representing a separate species. None of the isolates were found to produce ochratoxin A, kotanins, funalenone or pyranonigrins. The novel species was most closely related to A. niger, and was isolated from soil from Brazil, Australia, USA and The Netherlands, and from grape berries from Portugal. The type strain of Aspergillus brasiliensis sp. nov. is CBS 101740(T) (=IMI 381727(T)=IBT 21946(T)).