Species Of Aspergillus 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 .

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.

Aflatoxigenic Aspergillus Modulates Aflatoxin-B1 Levels through an Antioxidative Mechanism.

Aflatoxins (AFs) are considered to play important functions in species of Aspergillus section Flavi including an antioxidative role, as a deterrent against fungivorous insects, and in antibiosis. Atoxigenic Flavi are known to degrade AF-B1 (B1). To better understand the purpose of AF degradation, we investigated the degradation of B1 and AF-G1 (G1) in an antioxidative role in Flavi. Atoxigenic and toxigenic Flavi were treated with artificial B1 and G1 with or without the antioxidant selenium (Se), which is expected to affect levels of AF. After incubations, AF levels were measured by HPLC. To estimate which population would likely be favoured between toxigenic and atoxigenic Flavi under Se, we investigated the fitness, by spore count, of the Flavi as a result of exposure to 0, 0.40, and 0.86 µg/g Se in 3%-sucrose cornmeal agar (3gCMA). Results showed that levels B1 in medium without Se were reduced in all isolates, while G1 did not significantly change. When the medium was treated with Se, toxigenic Flavi significantly digested less B1, while levels of G1 significantly increased. Se did not affect the digestion of B1 in atoxigenic Flavi, and also did not alter levels of G1. Furthermore, atoxigenic strains were significantly fitter than toxigenic strains at Se 0.86 µg/g 3gCMA. Findings show that while atoxigenic Flavi degraded B1, toxigenic Flavi modulated its levels through an antioxidative mechanism to levels less than they produced. Furthermore, B1 was preferred in the antioxidative role compared to G1 in the toxigenic isolates. The higher fitness of atoxigenic over toxigenic counterparts at a plant non-lethal dose of 0.86 µg/g would be a useful attribute for integration in the broader biocontrol prospects of toxigenic Flavi.

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.

Development of RFLP method for rapid differentiation of Aspergillus flavus and Aspergillus oryzae, two species with high importance in clinical and food microbiology.

Aspergillus flavus and Aspergillus oryzae, two species of Aspergillus section Flavi, are of utmost significance in health, medicine, biotechnology, and foods industries. The methods currently used in mycology for the discrimination of these two closely related species were unable to definitively and rapidly distinguish. The present study aimed to develop a restriction fragment length polymorphism (RFLP) test based on the cyp51A gene to discriminate between A. flavus and A. oryzae. The cyp51A gene sequences of A. flavus and A. oryzae reference strains were amplified using the specific primers CYP51AF and CYP51AR. The polymerase chain reaction (PCR) products were subjected to digestion with a restriction enzyme, HincII. The polymerase chain reaction (PCR)- RFLP test created specific patterns for standard strains, as well as clinical and environmental A. flavus and A. oryzae isolates. The one-enzyme PCR-RFLP test on the cyp51A gene designed in the present study is a remarkably simple, reliable, and low-cost method for the accurate and rapid differentiation of A. flavus and A. oryzae isolated from clinical and environmental samples.

Multicenter Study of Susceptibility of Aspergillus Species Isolated from Iranian University Hospitals to Seven Antifungal Agents.

ABSTRACTAspergillus species are a major cause of life-threatening invasive infections and noninvasive diseases. This study seeks to investigate the frequency of Aspergillus species among Iranian patients and their susceptibility to seven antifungals. In a cross-sectional study, 233 Aspergillus isolates were collected from 11 university hospitals in Iran between 2018 and 2021. Aspergillus isolates were identified based on colony morphology, microscopic characteristics, PCR-restriction fragment length polymorphism (RFLP), and sequencing of the beta-tubulin gene. The CLSI M38-A2 reference methodology was used for antifungal susceptibility testing of amphotericin B, voriconazole, posaconazole, itraconazole, luliconazole, isavuconazole, and caspofungin. Members of Aspergillus section Flavi (117/233, 50.2%), Aspergillus section Nigri (77/233, 33.1%), Aspergillus section Fumigati (21/233, 9%), Aspergillus section Terrei (14/233, 6%), Aspergillus pseudodeflectus (2/233, 0.85%), and Aspergillus melleus (2/233, 0.85%) were isolated from the samples. The lowest 0.25 MIC90 values for all isolates tested were for luliconazole (0.016 μg/mL) and isavuconazole (0.250 μg/mL), and the highest value was observed for itraconazole (≥ 8μg/mL). The 90% minimum effective concentration (MEC90) value for caspofungin was 0.125 μg/mL. MIC90 values for voriconazole, amphotericin B, and posaconazole were 1, 2, and 2 μg/mL, respectively. The non-wild-type species were presented for amphotericin B (3%), voriconazole (1.3%), posaconazole (2.6%), luliconazole (1.3%), isavuconazole (1.7%), and caspofungin (4.7%). Positive correlations in the MIC values of azole antifungals were observed, and using one azole increases the MIC value rates of other ones. None of the species were pan-azole resistant. Species of Aspergillus section Flavi were the most common Aspergillus species isolated from Iranian samples. Luliconazole, caspofungin, and isavuconazole present the most effective antifungal agents for treatment of infection due to Aspergillus species. Susceptibility tests should be performed frequently in each region for the best management of patients.IMPORTANCE Aspergillus species are the leading cause of invasive aspergillosis in immunocompromised hosts. The susceptibility of Aspergillus species to antifungal agents might be different. Azole-resistant species have emerged worldwide. Performing susceptibility testing in each region can help in the best management of patients. Here, we show the epidemiology and distribution of Aspergillus species in Iran and their susceptibility patterns for seven antifungal agents. The significant points of the present study are that species of Aspergillus section Flavi are the most prevalent Aspergillus species isolated from 11 university hospitals. Luliconazole, caspofungin, and isavuconazole were effective antifungal agents against all Aspergillus species.

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.

Low-cost, specific PCR assays to identify the main aflatoxigenic species of Aspergillus section Flavi

Aflatoxins are fungal metabolites that are present as contaminants in food globally. Most aflatoxigenic species belong to Aspergillus section Flavi, and the main ones are grouped in the A. flavus clade, where many cryptic species that are difficult to discriminate are found. In this study, we investigated inter- and intraspecific diversity of the A. flavus clade to develop low-cost, species-specific PCR assays for identifying aflatoxigenic species. A total of 269 sequences of the second largest subunit of RNA polymerase II (RPB2) locus were retrieved from GenBank, and primer pairs were designed using data mining to identify A. flavus, A. parasiticus, and A. novoparasiticus. Species-specific amplicons of approximately 620, 350, and 860 bp enabled identification of target species as A. flavus, A. parasiticus, and A. novoparasiticus, respectively.

A Molecular Networking Based Discovery of Diketopiperazine Heterodimers and Aspergillicins from Aspergillus caelatus.

The number of species in Aspergillus section Flavi has recently increased to 36 and includes some of the most important and well-known species in the genus Aspergillus. Numerous secondary metabolites, especially mycotoxins, have been reported from species such as A. flavus; however many of the more recently described species are less studied from a chemical point of view. This paper describes the use of MS/MS-based molecular networking to investigate the metabolome of A. caelatus leading to the discovery of several new diketopiperazine dimers and aspergillicins. An MS-guided isolation procedure yielded six new compounds, including asperazines D-H (1-5) and aspergillicin H (6). Asperazines G and H are artifacts derived from asperazines E and F formed during the separation process by formic acid. Two known compounds, aspergillicins A and C (7 and 8), were isolated from the same strain. Structures were elucidated by analyzing their HR-MS/MS and NMR spectroscopic data. The absolute configuration of asperazines D-F and aspergillicin H were deduced from the combination of NMR, Marfey's method, and ECD analyses.

Temperature Influences on Interactions Among Aflatoxigenic Species of Aspergillus Section Flavi During Maize Colonization.

Fungal species within Aspergillus section Flavi contaminate food and feed with aflatoxins. These toxic fungal metabolites compromise human and animal health and disrupt trade. Genotypically and phenotypically diverse species co-infect crops, but temporal and spatial variation in frequencies of different lineages suggests that environmental factors such as temperature may influence structure of aflatoxin-producing fungal communities. Furthermore, though most species within Aspergillus section Flavi produce sclerotia, divergent sclerotial morphologies (small or S-type sclerotia vs. large or L-type sclerotia) and differences in types and quantities of aflatoxins produced suggest lineages are adapted to different life strategies. Temperature is a key parameter influencing pre- and post-harvest aflatoxin contamination of crops. We tested the hypothesis that species of aflatoxin-producing fungi that differ in sclerotial morphology will vary in competitive ability and that outcomes of competition and aflatoxin production will be modulated by temperature. Paired competition experiments between highly aflatoxigenic S-type species (A. aflatoxiformans and Lethal Aflatoxicosis Fungus) and L-type species (A. flavus L morphotype and A. parasiticus) were conducted on maize kernels at 25 and 30°C. Proportions of each isolate growing within and sporulating on kernels were measured using quantitative pyrosequencing. At 30°C, S-type fungi were more effective at host colonization compared to L-type isolates. Total aflatoxins and the proportion of B vs. G aflatoxins were greater at 30°C compared to 25°C. Sporulation by L-type isolates was reduced during competition with S-type fungi at 30°C, while relative quantities of conidia produced by S-type species either increased or did not change during competition. Results indicate that both species interactions and temperature can shape population structure of Aspergillus section Flavi, with warmer temperatures favoring growth and dispersal of highly toxigenic species with S-type sclerotia.

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.

Genetic map and heritability of Aspergillus flavus

The carcinogenic aflatoxins are a human health concern as well as an economic burden to corn, peanut and other crops grown within the United States and globally. Aflatoxins are produced by fungi species in Aspergillus section Flavi, primarily Aspergillus flavus. Though previously thought of as only asexual, A. flavus has recently been found to undergo sexual reproduction both in laboratory crosses and in the field. To elucidate the consequences of genetic exchange through a single generation of the sexual cycle within A. flavus, we constructed genetic maps based on three mapping populations, each composed of the parental strains and approximately 70 F1 progeny. Genome-wide data using double digest Restriction Associated DNA sequencing identified 496, 811, and 576 significant polymorphisms differentiating parents across eight linkage groups; these polymorphisms served as markers. Average spacing between marker loci was 3.1, 2.1, and 3.5 map units and overall map length was 1504.4, 1669.2, and 2001.3 cM. Recombination was non-randomly distributed across chromosomes with an average rate of recombination of about 46.81 cM per Mbp. We showed inheritance of mitochondrial loci from the sclerotial (female) parent in crosses, whereas nuclear loci showed a 1:1 segregation ratio from both parents. The linkage map will be useful in QTL analyses to identify traits that increase sexual fertility in A. flavus and modulate aflatoxin production, both of which have significant implications for sustainable reduction of aflatoxin contamination using biological control agents.

Occurrence and toxigenic potential of Aspergillus section Flavi on wheat and sorghum silages in Uruguay

ABSTRACT Species belonging to Aspergillus section Flavi occur naturally in crops and can cause food spoilage and/or toxin production. The aim of this study was to determine the occurrence and diversity of the species of Aspergillus section Flavi found in wheat and sorghum at harvest time and during silage storage, and to evaluate the toxigenic potential of the isolates to determine the contamination risk of mycotoxins in grains. Strains from Aspergillus flavus and Aspergillus parasiticus were found based on multi-gene phylogenetic analyses. This is the first report on the presence of A. parasiticus in wheat from Uruguay. Of the 80 isolates Aspergillus section Flavi, 30% produced aflatoxins (AFs), mainly type B1, and 25% produced cyclopiazonic acid (CPA). Within the isolates from wheat samples, 35% were AFs producers and 27.5% were CPA producers. Among the Aspergillus section Flavi isolates from sorghum, 25% were AFs producers while 22.5% were CPA producers. This work contributes to the knowledge of the species in crops and helps define appropriate strategies for the prevention and control of contamination with AFs and CPA by Aspergillus section Flavi fungi.

Toxigenicity and phylogeny of Aspergillus section Flavi in poultry feed in Iran.

Background and Purpose:This study was conducted to evaluate the presence of aflatoxigenic strains and level of aflatoxin in poultry feed. Aflatoxigenic strains were investigated in corn and soybean meal as the ingredients of poultry feed, as well as in two types of commercial feed, namely pellet and mash. The gene sequencing was performed to identify the species of Aspergillus section Flavi.Materials and Methods:All samples were randomly collected from feed storage silos located in Iran in 2018. The samples were cultured on specialized media for 2 weeks at 28ºC. Identification of Aspergillus section Flavi isolates was based on macro- and microscopic morphological criteria and molecular analysis. The thin-layer chromatography (TLC) was applied to confirm the aflatoxigenic isolates. In addition, the level of aflatoxin B1 (AFB1) produced by these isolates was determined by high-performance liquid chromatography. The strains were subjected to sequence analysis, and Bt2 PCR products were purified by the QIAquick PCR purification kit. At the final stage, the phylogenetic tree was built.Results:Among 54 isolates identified as Aspergillus section Flavi, 20 (37%) isolates were found to produce aflatoxin at a range of 11.28±1.18 to 2239.92±92.26 µg/g fungal dry weight. The aflatoxigenic isolates had the frequencies of 45%, 40%, 10%, and 5% in the corn, pellet, soybean meal, and mash samples, respectively. Furthermore, the mean concentrations of AFB1 were significantly higher in the corn samples (707.04±39.05) than that of other poultry feed samples (P<0.05). A total of 34 (63%) isolates were detected as non-aflatoxigenic on the yeast extract-sucrose broth in TLC analysis. The toxigenic isolates produced the highest (2232.62±55.49) and lowest (11.28±1.18) levels of AFB1 in the corn samples, compared to other feedstuffs. Furthermore, the mean level of AFB1 in mash product was 554.09±10.36 µg/g, compared to a mean level of 229.22±11.09 µg/g in pellets. The isolates were randomly selected, sequenced, and then analyzed. Subsequently, the phylogenetic tree of Aspergillus section Flavi was plotted.Conclusion:The process of converting raw ingredients to compound poultry feed is more hazardous when there is not enough time and temperature provided to eliminate aflatoxigenic isolates. Therefore, Aspergillus section Flavi in poultry feed can pose a threat to the poultry industry and poultry products, thereby affecting the health status of humans. Unprocessed/processed materials, such as corns and pelleted feed, need further monitoring, especially when conditions are not optimal for destroying the fungus.

Polyphasic characterization of Aspergillus section Flavi isolated from animal feeds in Algeria

AbstractIn Algeria, little information is available on the population structure of Aspergillus section Flavi in raw materials and resultant animal feeds. A total of 172 isolates belonging to Aspergillus section Flavi were recovered from 57 animal feeds and identified on the basis of macro and micro‐morphological characters, mycotoxin production and genetic relatedness. For the molecular analysis, sequencing of the calmodulin gene (CaM) and the internal transcribed spacer (ITS) regions were performed for representative isolates. Four distinct morphotypes were distinguished: Aspergillus flavus (78.5%), Aspergillus tamarii (19.2%), Aspergillus parasiticus (1.7%), and Aspergillus alliaceus (0.6%). All A. flavus isolates were of the L type and no correlation between sclerotia production and aflatoxigenicity was observed. Our results showed that 68% of the A. flavus strains produced aflatoxins B (AFB), and 72.7% were cyclopiazonic acid (CPA) producers. The three isolates of A. parasiticus were able to produce AFB and aflatoxins G but not CPA whereas, all the strains of A. tamarii produced only CPA. The obtained results revealed the presence of different species of Aspergillus section Flavi, among which were aflatoxin producers. This study provides evidence useful for considerations in aflatoxin control strategies.

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.