Species Of Section Flavi Research Articles

Simultaneous detection of mycotoxigenic Aspergillus species of sections Circumdati and Flavi using multiplex digital PCR.

Populations of ochratoxin-producing Aspergillus section Circumdati species and aflatoxin-producing Aspergillus section Flavi species frequently coexist in soil and are the main sources of mycotoxin contamination of tree nuts. Identification of mycotoxigenic Aspergillus species in these sections is difficult using traditional isolation and culture methods. We developed a multiplex digital PCR (dPCR) assay to detect and quantify Aspergillus ochraceus, Aspergillus westerdijkiae, and Aspergillus steynii (section Circumdati), as well as Aspergillus flavus and Aspergillus parasiticus (section Flavi), in environmental samples based on species-specific calmodulin gene sequences. Relative quantification of each species by dPCR of mixed-species templates correlated with corresponding DNA input ratios. Target species could be detected in soil inoculated with conidia from each species. Non-target species of sections Circumdati, Flavi, and Nigri were generally not detectable using this dPCR method. Detected non-target species (Aspergillus fresenii, Aspergillus melleus, Aspergillus sclerotiorum, and Aspergillus subramanianii) were discernible from A. ochraceus in dual-template dPCR reactions based on differential fluorescence intensity.

Crop Host Influences on Outcomes of Competition Between Aspergillus Section Flavi Species Coinfecting Maize and Groundnuts

Aspergillus section Flavi species co-occur and contaminate crops, including maize and groundnuts, with aflatoxins. Competition among A. flavus genotypes is influenced by crop host, but competition between Aspergillus species has not been examined. The objectives of the current study were to (i) assess competition among four aflatoxin-producing species on maize and groundnuts and (ii) evaluate within-species variation in competitive ability during coinfection with another species on the two crops. For the first objective, maize and groundnut kernels were coinoculated with all possible pairs of A. flavus, A. parasiticus, A. aflatoxiformans, and an unnamed taxon known as the Lethal Aflatoxicosis Fungus (LAF). For the second objective, three isolates from each of the four species were coinoculated with a representative isolate of a competing species on the two hosts. In all experiments, isolates were cocultured for 7 days at 30°C, and then aflatoxins and total conidia were measured, and the percentages of each species within a treatment were assessed with quantitative pyrosequencing. Maize kernels supported greater aflatoxin production than groundnuts, whereas groundnuts supported greater sporulation than maize. Hosts differentially influenced competition between species, with A. flavus generally more competitive on maize and LAF more competitive on groundnuts. Overall, A. flavus and LAF were the most competitive species, whereas A. parasiticus was the least competitive. However, isolates within a species varied in competitive ability and in their response to host and competing species. Results suggest that although crop hosts influence Aspergillus community composition, within-species variability makes it difficult to predict outcomes of competition on a particular crop. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Assessment of early harvest in the prevention of aflatoxins in peanuts during drought stress conditions

The present study aimed to evaluate the effectiveness of early harvest in preventing aflatoxins in peanuts under drought-stress conditions. A field experiment was conducted on the 2018–2019 and 2019–2020 growing seasons in a greenhouse with an irrigation system to induce three drought stress conditions: no stress, mild, and severe stress. In addition, three harvest dates were proposed: two weeks earlier, one week earlier, and ideal harvest time. The mean peanut yield was 2634 kg/ha, considering the two growing seasons, and the drought stress conditions and harvest dates did not influence significantly. The shelling percentage was significantly higher in samples harvested at ideal harvest (77.7 %) than two weeks earlier (76.2 %) and was not influenced by drought stress conditions. Although a low mean percentage of grains with insect damage was identified, this percentage was statistically higher under severe stress (0.4 %) compared to no-stress conditions (0.2 %). The soil contamination ranged from 2.52 × 103 to 1.64 × 104 CFU/g of Aspergillus section Flavi, and the drought stress resulted in significantly higher concentrations in mild and severe stressed samples. A. section Flavi was found to infect all the peanut kernel samples. The drought stress resulted in higher percentages of A. section Flavi infections in samples from mild and severe stress conditions. The harvest date did not influence the soil and peanut kernel occurrence of A. section Flavi. A total of 435 and 796 strains of A. section Flavi were isolated from soil and peanut kernels, respectively. The potential of aflatoxin production by soil isolates was 31, 44, and 25 % for aflatoxin non-producers, aflatoxin B producers, and aflatoxin B and G producers, respectively, while in peanut kernel isolates were 44, 44, and 12 %. Three different A. section Flavi species were identified from peanut kernels: A. flavus, A. parasiticus, and A. pseudocaelatus. The mean aflatoxin concentration in peanut kernels was 42, 316, and 695.5 μg/kg in samples under no stress, mild stress, and severe stress conditions, respectively. Considering the harvest time, the mean aflatoxin concentration was 9.9, 334.3, and 614.2 μg/kg in samples harvested two weeks earlier, one week earlier, and in ideal harvest, respectively. In conclusion, the early harvest proved to be a viable, cost-free alternative for controlling aflatoxin in the peanut pre-harvest, resulting in a safer product and a better quality for sale and economic gain.

Complete genome of the toxic mold Aspergillus pseudotamarii isolate NRRL 25517 reveals genomic instability of the aflatoxin biosynthesis cluster.

Fungi can synthesize a broad array of secondary metabolite chemicals. The genes underpinning their biosynthesis are typically arranged in tightly linked clusters in the genome. For example, ∼25 genes responsible for the biosynthesis of carcinogenic aflatoxins by Aspergillus section Flavi species are grouped in a ∼70 Kb cluster. Assembly fragmentation prevents assessment of the role of structural genomic variation in secondary metabolite evolution in this clade. More comprehensive analyses of secondary metabolite evolution will be possible by working with more complete and accurate genomes of taxonomically diverse Aspergillus species. Here, we combined short- and long-read DNA sequencing to generate a highly contiguous genome of the aflatoxigenic fungus, Aspergillus pseudotamarii (isolate NRRL 25517 = CBS 766.97; scaffold N50 = 5.5 Mb). The nuclear genome is 39.4 Mb, encompassing 12,639 putative protein-encoding genes and 74-97 candidate secondary metabolite biosynthesis gene clusters. The circular mitogenome is 29.7 Kb and contains 14 protein-encoding genes that are highly conserved across the genus. This highly contiguous A. pseudotamarii genome assembly enables comparisons of genomic rearrangements between Aspergillus section Flavi series Kitamyces and series Flavi. Although the aflatoxin biosynthesis gene cluster of A. pseudotamarii is conserved with Aspergillus flavus, the cluster has an inverted orientation relative to the telomere and occurs on a different chromosome.

Identification of Green Coffee Contaminated Microfungi of the Genus <i>Aspergillus</i> on the Basis of Polyphasic Approach

The Aspergillus species are widespread in the environment, able to grow at high temperatures and minimal humidity, including in regions with a hot tropical climate. Some species have the potential to produce toxins. This causes the risk of contamination by fungi of the genus Aspergillus and the mycotoxins (MT) produced by them of plant materials and food products, which is possible at any stage of production, transportation and storage. In the volume of coffee imported to the Russian Federation, 85% is accounted for by raw materials (green coffee), for which the risks of mold damage remain at all stages preceding the roasting stage. It is relevant to study the species composition and toxinogenic properties of Aspergillus spp., which contaminate food raw materials for the production of mass consumption foodstuffs. Contaminated products include coffee, which is one of the basic products of the consumer basket. Reliable data on species identification and toxigenic potential of Aspergillus spp. can be obtained only with an integrated approach based on polyphasic taxonomy. The purpose of this work is to study the species composition of fungi of the genus Aspergillus isolated from green coffee using an integrated approach based on polyphasic taxonomy. The species composition of fungi of the genus Aspergillus from the internal mycoflora of 16 samples of green coffee beans of Arabica and Robusta. The species belonging of the isolated 34 singlspore isolates of Aspergillus spp. was determined by cultural and morphological methods and confirmed by molecular genetic analysis, i.e., RT-PCR with DNA markers of conservative sequences (ITS, CaM, β-tub), studied in vitro profile of produced secondary toxic metabolites. The dominance of species of the Niger section was established (A. niger, 90%, and A. tubingensis, A. carbonarius); then, in decreasing order, the species of section Flavi followed (A. flavus, 100%), sections Circumdati (A. ochraceus, 40% and A. westerdijkiae, 60%). In section Fumigati there was one strain of A. fumigatus. Analysis of the profile of toxic metabolites by HPLC-MS/MS in the multi-detection mode showed the production of mycotoxins by the following species: A. niger – fumonisin B2 and ochratoxin A, A. flavus – aflatoxins B1 and B2 together with sterigmatocystin, A. westerdijkiae – ochratoxin A and penicillic acid, A. ochraceus – penicillic acid. Amounts of produced MT show a high toxinogenic potential of Aspergillus spp. Thus, 20 out of 34 strains produced significant amounts of dangerous, regulated mycotoxins: AFL B1, OTA, FB2. Non-toxinogenic isolates were represented by the species A. niger, A. carbonarius, A. tubingensis, A. flavus, and A. fumigatus. A study of the species composition and toxinogenic properties of green coffee contaminants of the genus Aspergillus using a polyphasic approach was carried out in Russia for the first time.

A Simple CRISPR/Cas9 System for Efficiently Targeting Genes of Aspergillus Section Flavi Species, Aspergillus nidulans, Aspergillus fumigatus, Aspergillus terreus, and Aspergillus niger.

For Aspergillus flavus, a pathogen of considerable economic and health concern, successful gene knockout work for more than a decade has relied nearly exclusively on using nonhomologous end-joining pathway (NHEJ)-deficient recipients via forced double-crossover recombination of homologous sequences. In this study, a simple CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease) genome editing system that gave extremely high (>95%) gene-targeting frequencies in A. flavus was developed. It contained a shortened Aspergillus nidulans AMA1 autonomously replicating sequence that maintained good transformation frequencies and Aspergillus oryzae ptrA as the selection marker for pyrithiamine resistance. Expression of the codon-optimized cas9 gene was driven by the A. nidulans gpdA promoter and trpC terminator. Expression of single guide RNA (sgRNA) cassettes was controlled by the A. flavus U6 promoter and terminator. The high transformation and gene-targeting frequencies of this system made generation of A. flavus gene knockouts with or without phenotypic changes effortless. Additionally, multiple-gene knockouts of A. flavus conidial pigment genes (olgA/copT/wA or olgA/yA/wA) were quickly generated by a sequential approach. Cotransforming sgRNA vectors targeting A. flavus kojA, yA, and wA gave 52%, 40%, and 8% of single-, double-, and triple-gene knockouts, respectively. The system was readily applicable to other section Flavi aspergilli (A. parasiticus, A. oryzae, A. sojae, A. nomius, A. bombycis, and A. pseudotamarii) with comparable transformation and gene-targeting efficiencies. Moreover, it gave satisfactory gene-targeting efficiencies (>90%) in A. nidulans (section Nidulantes), A. fumigatus (section Fumigati), A. terreus (section Terrei), and A. niger (section Nigri). It likely will have a broad application in aspergilli. IMPORTANCE CRISPR/Cas9 genome editing systems have been developed for many aspergilli. Reported gene-targeting efficiencies vary greatly and are dependent on delivery methods, repair mechanisms of induced double-stranded breaks, selection markers, and genetic backgrounds of transformation recipient strains. They are also mostly strain specific or species specific. This developed system is highly efficient and allows knocking out multiple genes in A. flavus efficiently either by sequential transformation or by cotransformation of individual sgRNA vectors if desired. It is readily applicable to section Flavi species and aspergilli in other sections ("section" is a taxonomic rank between genus and species). This cross-Aspergillus section system is for wild-type isolates and does not require homologous donor DNAs to be added, NHEJ-deficient strains to be created, or forced recycling of knockout recipients to be performed for multiple-gene targeting. Hence, it simplifies and expedites the gene-targeting process significantly.

Impact of frequency of application on the long-term efficacy of the biocontrol product Aflasafe in reducing aflatoxin contamination in maize.

Aflatoxins, produced by several Aspergillus section Flavi species in various crops, are a significant public health risk and a barrier to trade and development. In sub-Saharan Africa, maize and groundnut are particularly vulnerable to aflatoxin contamination. Aflasafe, a registered aflatoxin biocontrol product, utilizes atoxigenic A. flavus genotypes native to Nigeria to displace aflatoxin producers and mitigate aflatoxin contamination. Aflasafe was evaluated in farmers' fields for 3 years, under various regimens, to quantify carry-over of the biocontrol active ingredient genotypes. Nine maize fields were each treated either continuously for 3 years, the first two successive years, in year 1 and year 3, or once during the first year. For each treated field, a nearby untreated field was monitored. Aflatoxins were quantified in grain at harvest and after simulated poor storage. Biocontrol efficacy and frequencies of the active ingredient genotypes decreased in the absence of annual treatment. Maize treated consecutively for 2 or 3 years had significantly (p < 0.05) less aflatoxin (92% less) in grain at harvest than untreated maize. Maize grain from treated fields subjected to simulated poor storage had significantly less (p < 0.05) aflatoxin than grain from untreated fields, regardless of application regimen. Active ingredients occurred at higher frequencies in soil and grain from treated fields than from untreated fields. The incidence of active ingredients recovered in soil was significantly correlated (r = 0.898; p < 0.001) with the incidence of active ingredients in grain, which in turn was also significantly correlated (r = -0.621, p = 0.02) with aflatoxin concentration. Although there were carry-over effects, caution should be taken when drawing recommendations about discontinuing biocontrol use. Cost-benefit analyses of single season and carry-over influences are needed to optimize use by communities of smallholder farmers in sub-Saharan Africa.

Small NRPS-like enzymes in Aspergillus sections Flavi and Circumdati selectively form substituted pyrazinone metabolites.

Aspergillus fungi produce mycotoxins that are detrimental to human and animal health. Two sections of aspergilli are of particular importance to cereal food crops such as corn and barley. Aspergillus section Flavi species like A. flavus and A. parasiticus produce aflatoxins, while section Circumdati species like A. ochraceus and A. sclerotiorum produce ochratoxin A. Mitigating these toxins in food and feed is a critical and ongoing worldwide effort. We have previously investigated biosynthetic gene clusters in Aspergillus flavus that are linked to fungal virulence in corn. We found that one such cluster, asa, is responsible for the production of aspergillic acid, an iron-binding, hydroxamic acid-containing pyrazinone metabolite. Furthermore, we found that the asa gene cluster is present in many other aflatoxin- and ochratoxin-producing aspergilli. The core gene in the asa cluster encodes the small nonribosomal peptide synthetase-like (NRPS-like) protein AsaC. We have swapped the asaC ortholog from A. sclerotiorum into A. flavus, replacing its native copy, and have also cloned both asaC orthologs into Saccharomyces cerevisiae. We show that AsaC orthologs in section Flavi and section Circumdati, while only containing adenylation-thiolation-reductase (ATR) domains, can selectively biosynthesize distinct pyrazinone natural products: deoxyaspergillic acid and flavacol, respectively. Because pyrazinone natural products and the gene clusters responsible for their production are implicated in a variety of important microbe-host interactions, uncovering the function and selectivity of the enzymes involved could lead to strategies that ultimately benefit human health.

MORPHOCULTURAL CHARACTERIZATION AND QUALITATIVE ASSESSMENT OF AFLATOXINS PRODUCING Aspergillus flavus ASSOCIATED WITH Arachis hypogaea IN PUNJAB, PAKSITAN

Aflatoxins, produced by ubiquitous Aspergillus section Flavi species, are polyketide pathway-derived secondary metabolites and, due to their highly carcinogenic, mutagenic, and teratogenic nature, pose human severe health issues globally. In this study, raw groundnut kernels were collected from groundnut growing districts of Punjab, Pakistan, at pre and post-harvest stages. Groundnut-associated Aspergillus flavus isolates were morphologically identified, and their respective aflatoxins producing potential was determined through cultural methods; UV illumination and ammonia vapor exposure. Three hundred isolates were cultured on differential media, Malt extract agar, and Sabouraud dextrose agar, for studying typical morphocultural characteristics, and detailed microscopy. Observed phenotypic variability assorted isolates into twenty groups. Representative isolate of each group was grown on coconut agar medium and exposed to UV illumination and ammonia vapors to observe possible fluorescence excitation and reverse colony color change, respectively. Results of both cultural methods conformed to each other; 35% of isolates produced fluorescent ring upon UV exposure and pinkish reverse colony color upon ammonia vapors exposure (toxigenic), 50% isolates showed no change (atoxigenic), and 15 % showed unclear results. This study provides evidence that groundnut kernels are highly exposed to toxigenic A. flavus strains both at pre and post-harvest stages.

Efficacy of sodium metabisulphite for control of Aspergillus flavus and aflatoxin B1 contamination in vitro and in chilli powder and whole red chillies

Chillies are an important spice in many regions of the world. During the production and processing phases, they are prone to infection by mycotoxigenic fungi, especially Aspergillus Section Flavi species and contamination with aflatoxins. There is significant interest in controlling aflatoxin B1 (AFB1) contamination in such spices to ensure they remain below the legislative limits for human consumption. This study initially examined the potential efficacy of sodium metabisulphite (NaMBS, up to 5000 mg/L) for the control of growth and AFB1 production by Aspergillus flavus strains in vitro on a chilli-based medium modified to 0.93–0.995 water activity, aw). Subsequent studies examined efficacy of NaMBS in stored naturally contaminated chilli powder and whole red chillies, or when inoculated with A. flavus at 0.70, 0.80, 0.90, 0.95 aw and 30 °C for 20 days. NaMBS was effective in vitro in controlling growth of the three strains of A. flavus at the different aw levels examined. No growth occurred with >500–1250 NaMBS mg/L. AFB1 was only produced at 0.98 aw with complete inhibition at ≥1250 mg/L of the preservative at 30 °C. The in vitro ED50, ED90 for NaMBS varied with aw conditions. In situ studies with chilli powder and whole red dry chillies (naturally contaminated or + conidial inoculum of A. flavus) showed that at 0.70 and 0.80 aw, NaMBS treatments of 1000 and 2000 mg/L resulted in AFB1 contamination levels below the legislative limits for spices. However, under wetter conditions at 0.90 and 0.95 aw, AFB1 contamination of stored chilli powder, −/+ A. flavus inoculum, were above the legislative limits, even with 2000 mg/L NaMBS and 20 days storage at 30 °C. Stored whole chillies inoculated with A. flavus resulted in a reduction of populations, especially at 2000 mg/L NaMBS. Control of AFB1 was achieved only with 2000 mg/L NaMBS treatment after 10 and 20 days storage. Studies with commercial laminated sheets containing immobilised NaMBS with slow release of SO2 properties significantly reduced fungal populations and effectively controlled AFB1 contamination of the stored and packaged whole red chillies.

Mass Spectrometry-Based Network Analysis Reveals New Insights Into the Chemodiversity of 28 Species in Aspergillus section Flavi.

Aspergillus section Flavi includes some of the most famous mycotoxin producing filamentous fungi known to mankind. In recent years a number of new species have been included in section Flavi, however these species have been much less studied from a chemical point of view. In this study, we explored one representative strain of a total of 28 fungal species in section Flavi by systematically evaluating the relationship between taxonomy and secondary metabolites with LC-MS/MS analysis for the first time and dereplication through an in-house database and the Global Natural Product Social Molecular Networking (GNPS) platform. This approach allowed rapid identification of two new cyclopiazonic acid producers (A. alliaceus and A. arachidicola) and two new tenuazonic acid producers (A. arachidicola and A. leporis). Moreover, for the first time we report species from section Flavi to produce fumifungin and sphingofungins B-D. Altogether, this study emphasizes that the chemical diversity of species in genus Aspergillus section Flavi is larger than previously recognized, and especially that understudied species are prolific producers of important mycotoxins such as fumi- and sphingofungins not previously reported from this section. Furthermore, our work demonstrates Global Natural Product Social (GNPS) Molecular Networking as a powerful tool for large-scale chemotaxonomic analysis of closely related species in filamentous fungi.

Determining future aflatoxin contamination risk scenarios for corn in Southern Georgia, USA using spatio-temporal modelling and future climate simulations

Aflatoxins (AFs) are produced by fungi in crops and can cause liver cancer. Permitted levels are legislated and batches of grain are rejected based on average concentrations. Corn grown in Southern Georgia (GA), USA, which experiences drought during the mid-silk growth period in June, is particularly susceptible to infection by Aspergillus section Flavi species which produce AFs. Previous studies showed strong association between AFs and June weather. Risk factors were developed: June maximum temperatures > 33 °C and June rainfall < 50 mm, the 30-year normals for the region. Future climate data were estimated for each year (2000–2100) and county in southern GA using the RCP 4.5 and RCP 8.5 emissions scenarios. The number of counties with June maximum temperatures > 33 °C and rainfall < 50 mm increased and then plateaued for both emissions scenarios. The percentage of years thresholds were exceeded was greater for RCP 8.5 than RCP 4.5. The spatial distribution of high-risk counties changed over time. Results suggest corn growth distribution should be changed or adaptation strategies employed like planting resistant varieties, irrigating and planting earlier. There were significantly more counties exceeding thresholds in 2010–2040 compared to 2000–2030 suggesting that adaptation strategies should be employed as soon as possible.

A Recent Overview of Producers and Important Dietary Sources of Aflatoxins.

Aflatoxins (AFs) are some of the most agriculturally important and harmful mycotoxins. At least 20 AFs have been identified to this date. Aflatoxin B1 (AFB1), the most potent fungal toxin, can cause toxicity in many species, including humans. AFs are produced by 22 species of Aspergillus section Flavi, 4 species of A. section Nidulantes, and 2 species of A. section Ochraceorosei. The most important and well-known AF-producing species of section Flavi are Aspergillus flavus, A. parasiticus, and A. nomius. AFs contaminate a wide range of crops (mainly groundnuts, pistachio nuts, dried figs, hazelnuts, spices, almonds, rice, melon seeds, Brazil nuts, and maize). Foods of animal origin (milk and animal tissues) are less likely contributors to human AF exposure. Despite the efforts to mitigate the AF concentrations in foods, and thus enhance food safety, AFs continue to be present, even at high levels. AFs thus remain a current and continuously pressing problem in the world.

A comparative genomics study of 23 Aspergillus species from section Flavi

Section Flavi encompasses both harmful and beneficial Aspergillus species, such as Aspergillus oryzae, used in food fermentation and enzyme production, and Aspergillus flavus, food spoiler and mycotoxin producer. Here, we sequence 19 genomes spanning section Flavi and compare 31 fungal genomes including 23 Flavi species. We reassess their phylogenetic relationships and show that the closest relative of A. oryzae is not A. flavus, but A. minisclerotigenes or A. aflatoxiformans and identify high genome diversity, especially in sub-telomeric regions. We predict abundant CAZymes (598 per species) and prolific secondary metabolite gene clusters (73 per species) in section Flavi. However, the observed phenotypes (growth characteristics, polysaccharide degradation) do not necessarily correlate with inferences made from the predicted CAZyme content. Our work, including genomic analyses, phenotypic assays, and identification of secondary metabolites, highlights the genetic and metabolic diversity within section Flavi.

Application of MALDI-TOF MS to species complex differentiation and strain typing of food related fungi: Case studies with Aspergillus section Flavi species and Penicillium roqueforti isolates

Filamentous fungi are one of the main causes of food losses worldwide and their ability to produce mycotoxins represents a hazard for human health. Their correct and rapid identification is thus crucial to manage food safety. In recent years, MALDI-TOF emerged as a rapid and reliable tool for fungi identification and was applied to typing of bacteria and yeasts, but few studies focused on filamentous fungal species complex differentiation and typing. Therefore, the aim of this study was to evaluate the use of MALDI-TOF to identify species of the Aspergillus section Flavi, and to differentiate Penicillium roqueforti isolates from three distinct genetic populations. Spectra were acquired from 23 Aspergillus species and integrated into a database for which cross-validation led to more than 99% of correctly attributed spectra. For P. roqueforti, spectra were acquired from 63 strains and a two-step calibration procedure was applied before database construction. Cross-validation and external validation respectively led to 94% and 95% of spectra attributed to the right population. Results obtained here suggested very good agreement between spectral and genetic data analysis for both Aspergillus species and P. roqueforti, demonstrating MALDI-TOF applicability as a fast and easy alternative to molecular techniques for species complex differentiation and strain typing of filamentous fungi.

The aspergillic acid biosynthetic gene cluster predicts neoaspergillic acid production inAspergillus sectionCircumdati

The fungusAspergillus flavus is an opportunistic crop pathogen that produces aflatoxins. Aflatoxins are potent carcinogenic and hepatotoxic secondary metabolites that are highly regulated in most countries.A. flavus also produces many other secondary metabolites and harbours more than 50 putative secondary metabolite biosynthetic gene clusters that have yet to be characterised. Bioactive secondary metabolites that augment the ability of the fungus to infect crops are of particular interest. Biosynthetic gene cluster 11 inA. flavus has been recently shown to encode for the biosynthesis of aspergillic acid, a toxic hydroxamic acid-containing pyrazinone compound that can bind iron, resulting in a red-orange pigment known as ferriaspergillin. A decrease inA. flavus pathogenicity and aflatoxin contamination was observed when aspergillic acid biosynthesis was blocked during maize seed infection. In this study, we probe the available genomes ofAspergillus species for biosynthetic gene cluster 11 homologs. We find that all species possessing gene cluster 11 produce aspergillic acid or a closely related isomer. We demonstrate that theAspergillus sectionFlavi species harbouring biosynthetic gene cluster 11 produce a mixture of aspergillic acid, hydroxyaspergillic acid, and aspergillic acid analogs differing only in the amino acid precursors. Interestingly, manyAspergillus sectionCircumdati species, known mainly for their production of the problematic mycotoxin ochratoxin A, also harbour gene cluster 11 homologs, but do not produce aspergillic acid. Instead, these species produce neoaspergillic acid and its hydroxylated analog neohydroxyaspergillic acid, indicating that cluster 11 is responsible for neoaspergillic acid biosynthesis inAspergillus sectionCircumdati.

Secondary Metabolite Dereplication and Phylogenetic Analysis Identify Various Emerging Mycotoxins and Reveal the High Intra-Species Diversity in Aspergillus flavus.

Aspergillus flavus is one of the most important mycotoxigenic species from the genus Aspergillus, due to its ability to synthesize the potent hepatocarcinogen, aflatoxin B1. Moreover, this fungus is capable of producing several other toxic metabolites from the class of indole-tetramates, non-ribosomal peptides, and indole-diterpenoids. Populations of A. flavus are characterized by considerable diversity in terms of morphological, functional and genetic features. Although for many years A. flavus was considered an asexual fungus, researchers have shown evidence that at best these fungi can exhibit a predominantly asexual existence. We now know that A. flavus contains functional genes for mating, uncovering sexuality as potential contributor for its diversification. Based on our results, we reconfirm that A. flavus is a predominant producer of B-type aflatoxins. Moreover, this fungus can decisively produce AFM1 and AFM2. We did not observe any clear relationship between mating-type genes and particular class of metabolites, probably other parameters such as sexual/asexual ratio should be investigated. A dynamic secondary metabolism was found also in strains intended to be used as biocontrol agents. In addition we succeeded to provide mass spectrometry fragmentation spectra for the most important classes of A. flavus metabolites, which will serve as identification cards for future studies. Both, metabolic and phylogenetic analysis proved a high intra-species diversity for A. flavus. These findings contribute to our understanding about the diversity of Aspergillus section Flavi species, raising the necessity for polyphasic approaches (morphological, metabolic, genetic, etc.) when dealing with this type of complex group of species.

Antifungal activity of silver nanoparticles and simvastatin against toxigenic species of Aspergillus

Aspergillus spp. are ubiquitous fungi that grow on stored grains. Some species produce toxins that can harm human and animal health, leading to hepato- and nephrotoxicity, immunosuppression and carcinogenicity. Major fungicides used to prevent fungal growth may be toxic to humans and their repeated use over time increases levels of resistance by microorganisms. Nanotechnology is an emerging field that allows use of antimicrobial compounds in a more efficient manner. In this study, was evaluated the antifungal activity of biogenic silver nanoparticles (AgNPs, synthesized by fungi) and simvastatin (SIM, a semi-synthetic drug), alone and in combination against three toxigenic species belonging to the genera Aspergillus section Flavi (Aspergillus flavus, Aspergillus nomius and Aspergillus. parasiticus) and two of section Circumdati (Aspergillus ochraceus and Aspergillus melleus). SIM exhibited a MIC50 of 78 μg/mL against species of Section Flavi and a MIC50 of 19.5 μg/mL against species of Section Circumdati. The MIC50 of AgNPs against Aspergillus flavus, Aspergillus nomius and Aspergillus parasiticus was 8 μg/mL, while the MIC50 was 4 μg/mL against Aspergillus melleus and Aspergillus ochraceus. Checkerboard assay showed that these compounds, used alone and in combination, have synergistic and additive effects against toxicogenic species of Aspergillus. Analysis by SEM gives an idea of the effect of SIM and AgNPs alone and in combination on spore germination and vegetative growth. Ultrastructural analysis revealed that spore germination was prevented, or aberrant hyphae were formed with multilateral branches upon treatment with SIM and AgNPs. These results reveal potential benefits of using combination of AgNPs and SIM to control fungal growth.

Aflatoxin Contamination of Dried Insects and Fish in Zambia

Dried insects and fish are important sources of income and dietary protein in Zambia. Some aflatoxin-producing fungi are entomopathogenic and also colonize insects and fish after harvest and processing. Aflatoxins are carcinogenic, immune-suppressing mycotoxins that are frequent food contaminants worldwide. Several species within Aspergillus section Flavi have been implicated as causal agents of aflatoxin contamination of crops in Africa. However, aflatoxin producers associated with dried fish and edible insects in Zambia remain unknown, and aflatoxin concentrations in these foods have been inadequately evaluated. The current study sought to address these data gaps to assess potential human vulnerability through the dried fish and edible insect routes of aflatoxin exposure. Caterpillars ( n = 97), termites ( n = 4), and dried fish ( n = 66) sampled in 2016 and 2017 were assayed for aflatoxin by using lateral flow immunochromatography. Average aflatoxin concentrations exceeded regulatory limits for Zambia (10 μg/kg) in the moth Gynanisa maja (11 μg/kg), the moth Gonimbrasia zambesina (Walker) (12 μg/kg), and the termite Macrotermes falciger (Gerstacker) (24 μg/kg). When samples were subjected to simulated poor storage, aflatoxins increased ( P < 0.001) to unsafe levels in caterpillars (mean, 4,800 μg/kg) and fish ( Oreochromis) (mean, 23 μg/kg). The L strain morphotype of A. flavus was the most common aflatoxin producer on dried fish (88% of Aspergillus section Flavi), termites (68%), and caterpillars (61%), with the exception of Gynanisa maja, for which A. parasiticus was the most common (44%). Dried fish and insects supported growth (mean, 1.3 × 109 CFU/g) and aflatoxin production (mean, 63,620 μg/kg) by previously characterized toxigenic Aspergillus section Flavi species, although the extent of growth and aflatoxigenicity depended on specific fungus-host combinations. The current study shows the need for proper storage and testing of dried insects and fish before consumption as measures to mitigate human exposure to aflatoxins through consumption in Zambia.

Taxonomy of Aspergillus section Flavi and their production of aflatoxins, ochratoxins and other mycotoxins.

Aflatoxins and ochratoxins are among the most important mycotoxins of all and producers of both types of mycotoxins are present in Aspergillus section Flavi, albeit never in the same species. Some of the most efficient producers of aflatoxins and ochratoxins have not been described yet. Using a polyphasic approach combining phenotype, physiology, sequence and extrolite data, we describe here eight new species in section Flavi. Phylogenetically, section Flavi is split in eight clades and the section currently contains 33 species. Two species only produce aflatoxin B1 and B2 (A. pseudotamarii and A. togoensis), and 14 species are able to produce aflatoxin B1, B2, G1 and G2: three newly described species A. aflatoxiformans, A. austwickii and A. cerealis in addition to A. arachidicola, A. minisclerotigenes, A. mottae, A. luteovirescens (formerly A. bombycis), A. nomius, A. novoparasiticus, A. parasiticus, A. pseudocaelatus, A. pseudonomius, A. sergii and A. transmontanensis. It is generally accepted that A. flavus is unable to produce type G aflatoxins, but here we report on Korean strains that also produce aflatoxin G1 and G2. One strain of A. bertholletius can produce the immediate aflatoxin precursor 3-O-methylsterigmatocystin, and one strain of Aspergillus sojae and two strains of Aspergillus alliaceus produced versicolorins. Strains of the domesticated forms of A. flavus and A. parasiticus, A. oryzae and A. sojae, respectively, lost their ability to produce aflatoxins, and from the remaining phylogenetically closely related species (belonging to the A. flavus-, A. tamarii-, A. bertholletius- and A. nomius-clades), only A. caelatus, A. subflavus and A. tamarii are unable to produce aflatoxins. With exception of A. togoensis in the A. coremiiformis-clade, all species in the phylogenetically more distant clades (A. alliaceus-, A. coremiiformis-, A. leporis- and A. avenaceus-clade) are unable to produce aflatoxins. Three out of the four species in the A. alliaceus-clade can produce the mycotoxin ochratoxin A: A. alliaceus s. str. and two new species described here as A. neoalliaceus and A. vandermerwei. Eight species produced the mycotoxin tenuazonic acid: A. bertholletius, A. caelatus, A. luteovirescens, A. nomius, A. pseudocaelatus, A. pseudonomius, A. pseudotamarii and A. tamarii while the related mycotoxin cyclopiazonic acid was produced by 13 species: A. aflatoxiformans, A. austwickii, A. bertholletius, A. cerealis, A. flavus, A. minisclerotigenes, A. mottae, A. oryzae, A. pipericola, A. pseudocaelatus, A. pseudotamarii, A. sergii and A. tamarii. Furthermore, A. hancockii produced speradine A, a compound related to cyclopiazonic acid. Selected A. aflatoxiformans, A. austwickii, A. cerealis, A. flavus, A. minisclerotigenes, A. pipericola and A. sergii strains produced small sclerotia containing the mycotoxin aflatrem. Kojic acid has been found in all species in section Flavi, except A. avenaceus and A. coremiiformis. Only six species in the section did not produce any known mycotoxins: A. aspearensis, A. coremiiformis, A. lanosus, A. leporis, A. sojae and A. subflavus. An overview of other small molecule extrolites produced in Aspergillus section Flavi is given.