Aspergillus Section Flavi Isolates Research Articles

A polyphasic characterisation of Aspergillus section Flavi isolated from Malaysian and Nigerian food grains and poultry feeds by phenotypic, chemotypic, and molecular methods

The taxonomy and toxigenicity of Aspergillus section Flavi (ASF) in various regions are still under-reported, as many potentially toxic species are continuously discovered. This study aims to determine the aflatoxigenicity and identify 74 isolates of ASF from Malaysian and Nigerian food grains and feeds using phenotypic, chemotypic, and molecular methods. Isolates were screened for sclerotium morphotype and aspergillic acid production phenotypically, while an ultra-fast reverse-phase high-performance liquid chromatography (HPLC) was optimised to quantify the cyclopiazonic acid and aflatoxins produced by the isolated ASF, based on which, they were characterised into phenotypes L-strains, S-strains, and NS-strains, and chemotypes I to VI. Chemotypic data and nucleotide sequences of β-tubulin and internal transcribed spacer (ITS) genes were utilised to identify the isolates’ species and determine their phylogenetic relationships. Aflatoxigenesis on solid media and multiplex polymerase chain reactions of four essential genes (aflR, aflM, aflD, and aflP) of aflatoxin biosynthesis were used to confirm the aflatoxigenicity of each isolate. All the six chemotypes of ASF were present among the isolated strains, with the aflatoxigenic chemotypes (55.41%) and CPA-positive isolates (68.92%) being significantly higher (p < 0.05) than the non-aflatoxigenic and CPA negative chemotypes. Aflatoxins and CPA were produced in the 0.25–19.58 ng/g range and 29.89–46.55 ng/g, respectively. Chemotypic data accurately differentiate toxigenic from atoxigenic species but only identify 90% of species names. Molecular data accurately resolved species but not toxigenicity. The results highlight the need for robust regulatory measures in the concerned regions to mitigate the risk of aflatoxicosis.

The Molecular Identification and Antifungal Susceptibility of Clinical Isolates of Aspergillus Section Flavi from Three French Hospitals.

(1) Background: Aspergillus flavus is a cosmopolitan mold with medical, veterinary, and agronomic concerns. Its morphological similarity to other cryptic species of the Flavi section requires molecular identification techniques that are not routinely performed. For clinical isolates of Aspergillus section Flavi, we present the molecular identification, susceptibility to six antifungal agents, and clinical context of source patients. (2) Methods: One hundred forty fungal clinical isolates were included in the study. These isolates, recovered over a 15-year period (2001-2015), were identified based on their morphological characteristics as belonging to section Flavi. After the subculture, sequencing of a part of the β-tubulin and calmodulin genes was performed, and resistance to azole antifungals was screened on agar plates containing itraconazole and voriconazole. Minimum inhibitory concentrations were determined for 120 isolates by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth microdilution method. (3) Results: Partial β-tubulin and calmodulin sequences analysis showed that 138/140 isolates were A. flavus sensu stricto, 1 isolate was A. parasiticus/sojae, and 1 was A. nomiae. Many of the isolates came from samples collected in the context of respiratory tract colonization. Among probable or proven aspergillosis, respiratory infections were the most frequent, followed by ENT infections. Antifungal susceptibility testing was available for isolates (n = 120, all A. flavus ss) from one hospital. The MIC range (geometric mean MIC) in mg/L was 0.5-8 (0.77), 0.5-8 (1.03), 0.125-2 (0.25), 0.03-2 (0.22), 0.25-8 (1.91), and 0.03-0.125 (0.061) for voriconazole, isavuconazole, itraconazole, posaconazole, amphotericin B, and caspofungin, respectively. Two (1.67%) isolates showed resistance to isavuconazole according to current EUCAST breakpoints with MICs at 8 mg/L for isavuconazole and voriconazole. One of these two isolates was also resistant to itraconazole with MIC at 2 mg/L. (4) Conclusions: The present characterization of a large collection of Aspergillus belonging to the Flavi section confirmed that A. flavus ss is the predominant species. It is mainly implicated in respiratory and ENT infections. The emergence of resistance highlights the need to perform susceptibility tests on section Flavi isolates.

OPTIMIZATION AND VALIDATION OF HPLC-FLD METHOD FOR DETERMINATION OF AFLATOXIN B1 AND PRODUCTION OF THIS MYCOTOXIN BY ASPERGILLUS SECTION FLAVI ISOLATES

A high performance liquid chromatography coupled with fluorescence detection (HPLC-FLD) was evaluated for the monitoring of aflatoxin B1 (AFB1) production by Aspergillus section Flavi isolates, which were found in the commodities of Slovak origin (barley and tomatoes). Rapid validation of the method was performed and the method was applied for the determination of the mycotoxin. Based on the calculated validation characteristics, the method showed high sensitivity and reproducibility. The LOD and LOQ determined were 0.03 and 0.10 µg.mL-1 which is fully acceptable for the determination of the AFB1 in isolates from plant commodities. The calibration graph was linear in the concentration range of 0.01– 10.0 µg.mL-1. The optimized method was used for the analysis of the mycotoxin in three Aspergillus section Flavi isolates. The submerse cultivation was performed at 25±1°C in yeast extract sucrose liquid medium and material was collected on the 7th, 14th and 21st day of cultivation. Simple sample extraction was carried out with use of ethyl acetate, and measurement was done with use of the mobile phase consisting of water and methanol by HPLC-FLD. Production of the AFB1 by isolates was ranging from &lt;LOD (0.03 µg. mL-1) to 49.38 µg.mL-1 with the highest concentration on the 14th day of cultivation. The maximum level of AFB1 was achieved by the Isolate 1 (isolated from barley, Kolíňany, 2018).

Characterization of 260 Isolates of Aspergillus Section Flavi Obtained from Sesame Seeds in Punjab, Pakistan.

Sesame Sesamum indicum L. is a major oil-based seed crop that has been widely cultivated and consumed in Pakistan. Unfortunately, sesame is highly prone to Aspergillus fungal growth in the field, and under inappropriate storage conditions can become contaminated with aflatoxins, the most potent carcinogen found in nature. Here, we have isolated a high number of Aspergillus isolates from sesame seeds in fresh and stored conditions obtained from rainfed and irrigated zones of Punjab, Pakistan, and characterized them for aflatoxigenic potentials. Using morphological identification techniques, 260 isolates were grouped as potential Aspergillus section Flavi, with 126 and 134 originating from the rainfed and irrigated zones, respectively. Out of 260 in total, 188 isolates were confirmed to produce aflatoxins. There were no significant differences in potential aflatoxigenic isolates with respect to the rainfed and irrigated zones. However, the number of potential aflatoxigenic isolates was significantly higher (p < 0.05) in stored samples than that of those from fresh sesame seeds in the rainfed and irrigated zone. Whole genome sequencing and comparative analyses of 12 select isolates have revealed that one of the A. flavus isolates, which produced very low aflatoxins (AFP10), has an elevated missense variant rate, numerous high impact mutations, and a 600 base pair deletion in the norB gene. In summary, our study provides insights into aflatoxigenic potential and the associated genetic diversity of indigenous Aspergillus section Flavi isolates and potential management strategies for reducing aflatoxin contamination levels in a major crop consumed in Punjab, Pakistan.

Aspergillus Section Flavi, Need for a Robust Taxonomy.

In a recent letter to the editor, Houbraken et al. (23) provide a series of recommendations to the microbiological community to prevent the taxonomic misidentification of genome-sequenced fungal strains. In the era of genomics and bioinformatics, postulating that 1 nucleotide (nt) within a gene can “correctly” identify a species does not seem plausible. However, the authors of the letter call this the “calmodulin barcode,” meaning nucleotide substitutions within a 506-nt region of the calmodulin gene (1). After the evolutionarily conserved rRNA (18S rRNA, internal transcribed spacer [ITS], 28S rRNA) and RNA polymerase II (2–4) showed no differences between Aspergillus flavus S- and L-morphotypes, attention shifted toward the calmodulin gene. Thus, without sequencing 18S rRNA, 28S rRNA, or the largest RNA polymerase II subunit, at least 34 new species of Aspergillus were named by Houbraken, Frisvad, Visagie, and coworkers (1, 5, 6). However, in a phylogenetic tree of 152 Aspergillus section Flavi isolates using the calmodulin 506-nt region, 40 Aspergillus minisclerotigenes isolates had only two nucleotide substitutions in common, namely, 100C>A and 269A>G, both of which are silent mutations (Fig. 1). However, only 269A>G discriminates A. minisclerotigenes from A. flavus, since 100C>A is present in three A. flavus isolates (GenBank accession numbers MK451387, MK451365, and MG517986) identified by the authors of the letter. We all agree that species identification is important; paradoxically, the calmodulin barcode assigns species based on a single-nucleotide polymorphism (SNP), while there are between 133,000 and 179,000 SNPs within A. flavus S- and L-morphotypes, respectively (7).

Climatic conditions and farm practices affected the prevalence of Aspergillus section Flavi on different types of dairy goat's feed

The present work aimed to determine the prevalence of aflatoxigenic Aspergillus section Flavi on different types of dairy goat's feed samples obtained from four dairy goat's farms around the central region of Peninsular Malaysia, and to examine the effects of climatic conditions (temperature, relative humidity) of the dairy goat's farms, and their feeding and storage practices on the fungal prevalence of different types of dairy goat's feed. A total of 60 goat's feed samples were obtained, and their proximate composition and water activity were determined, following which they were cultivated on DRBC and AFPA for total fungal load and Aspergillus section Flavi load determination, respectively. Fungal isolates were identified morphologically, and toxigenicity potentials of Aspergillus section Flavi isolates were determined using CCA. The temperature and relative humidity data of all farms were obtained from the Malaysian Meteorological Department. The total fungal loads (on DRBC) of the goat's feed samples were log 0.767 to 7.071 CFU/g which included the common feed contaminants such as Aspergillus, Fusarium, and Penicillium. The Aspergillus section Flavi loads (on AFPA) were log 0.667 to 3.206 CFU/g. Farm A yielded the highest number of Aspergillus section Flavi isolates as well as the highest number of aflatoxigenic isolates. It was found that climatic conditions and different practices between farms positively influenced the fungal prevalence on goat's feed samples based on the Pearson correlation analysis. The prevalence of mycotoxigenic isolates on goat's feed warrants for urgent intervention to ensure that goats are being fed with nutritionally adequate and safe feed. The presence of aflatoxigenic Aspergillus section Flavi isolates indicates the risk of aflatoxin B1 contamination on the goat's feed, aflatoxicosis development in the goats, and aflatoxin M1 bio-transformation in the goat's milk. This is a potential threat to the flourishing goat's milk industry in Malaysia.

Contamination of groundnut (Arachis hypogaea L.) with Aspergillus section Flavi communities and aflatoxin at the post-harvest stage

Groundnut (Arachis hypogaea L.) is essential to prepare numerous popular dishes across sub-Saharan Africa. However, groundnuts may contain hazardous aflatoxin levels. There is scant information on aflatoxin levels in Uganda and the causal agents of contamination in stored groundnut. High aflatoxin content hinders trading of groundnut and groundnut products in international markets, affecting Uganda's economy. The current study assessed contamination of groundnut with Aspergillus section Flavi fungi and aflatoxin under storage conditions. One hundred seventy-nine samples were sourced from farmers' household stores from 18 major groundnut-producing districts in seven agro-ecological zones (AEZ). Aflatoxin levels ranged from 0 to 1327 ng/g. Around 45% of the samples had aflatoxin levels above the Uganda National Bureau of Standards (10 ng/g). In addition, 1829 Aspergillus section Flavi isolates were recovered, and the A. flavus L morphotype dominated the communities (80%). Cropping systems, storage and climatic conditions in different AEZ influenced communities' structure composition and aflatoxin levels. There were significant (P < 0.05) differences in aflatoxin content and frequencies of Aspergillus species among AEZ and districts. The extremely high aflatoxin levels detected in samples from some districts beckoned the urgent need for management strategies are. Aflatoxin biocontrol products containing atoxigenic isolates of A. flavus, such as those detected in the study, could be used to reduce aflatoxin contamination in both field and storage conditions.

Occurrence of aflatoxin- and ochratoxin A-producing Aspergillus species in Thai dried chilli

The present study aimed to examine the presence of aflatoxin- and ochratoxin A (OTA)-producing fungi in dried chilli ( Capsicum spp.). A total of 120 chilli samples, including 60 samples each of chilli powder and dried chilli pods, were examined for fungal contamination. Results showed that 86.7% and 96.7% of chilli powder and dried chilli pods, respectively, were contaminated by fungi at a concentration of <10 3 CFU/g. Overall, 500 fungal isolates were recovered and identified using a morphological method. The most frequent strain was Aspergillus section Flavi (46.6%), followed by Aspergillus section Nigri (34%), Penicillium (7.6%), Aspergillus section Aspergillus (7.6%), Aspergillus section Circumdati (2.2%) and Rhizopus (2%). In total, 37.5% and 43.8% of Aspergillus section Flavi isolates obtained from dried chilli powder and dried chilli pods, respectively, produced aflatoxin. Of the 96 aflatoxigenic isolates, 96.9% of the strains produced aflatoxin B and 3.1% produced both aflatoxin B and G. However, β-tubulin sequence analysis revealed that all aflatoxin-producing isolates were identical to A. flavus . Moreover, 170 isolates of Aspergillus section Nigri , 11 of Aspergillus section Circumdati , 2 of Aspergillus section Flavi producing yellow-to-cinnamon colonies and 38 of Penicillium spp. were examined for their ability to produce OTA. In total, 1 isolate of Aspergillus section Nigri and 2 isolates of Aspergillus section Flavi produced OTA. The first isolate was identified as A. carbonarius , whereas the latter two isolates were both A. alliaceus . The highest OTA production by A. carbonarius (622.4 ng/g) was observed following incubation on MEA at 25 °C for 21 days. Conversely, the two A. alliaceus isolates produced OTA at levels of 583.7 and 743.7 ng/g, respectively, after cultivation on YES at 25 °C for 21 days. To the best of our knowledge, this is the first report regarding the natural occurrence of aflatoxin- and OTA-producing fungi in dried chilli from Thailand. • More than 95% of dried chilli powder and dried chilli pods collected in Thailand was contaminated with fungi. • Aspergillus section Flavi is the most common species in dried chilli. • Aspergillus section Flavi in dried chili samples is frequently aflatoxigenic strain. • Ochratoxin A producing species, A. carbonarius and A. alliaceus , were detected in dried chilli powder.

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.

A Polyphasic Approach to Compare the Genomic Profiles of Aflatoxigenic and Non-Aflatoxigenic Isolates of Aspergillus Section Flavi.

Aflatoxins (AF) are highly toxic compounds produced by Aspergillus section Flavi. They spoil food crops and present a serious global health hazard to humans and livestock. The aim of this study was to examine the phylogenetic relationships among aflatoxigenic and non-aflatoxigenic Aspergillus isolates. A polyphasic approach combining phylogenetic, sequence, and toxin analyses was applied to 40 Aspergillus section Flavi isolates collected from eight countries around the world (USA, Philippines, Egypt, India, Australia, Indonesia, China, and Uganda). This allows one to pinpoint the key genomic features that distinguish AF producing and non-producing isolates. Based on molecular identification, 32 (80%) were identified as A. flavus, three (7.5%) as A. parasiticus, three (7.5%) as A. nomius and one (2.5%) as A. tamarii. Toxin analysis showed that 22 (55%) Aspergillus isolates were aflatoxigenic. The majority of the toxic isolates (62.5%) originated from Egypt. The highest aflatoxin production potential was observed in an A. nomius isolate which is originally isolated from the Philippines. DNA-based molecular markers such as random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) were used to evaluate the genetic diversity and phylogenetic relationships among these 40 Aspergillus isolates, which were originally selected from 80 isolates. The percentage of polymorphic bands in three RAPD and three ISSR primers was 81.9% and 79.37%, respectively. Analysis of molecular variance showed significant diversity within the populations, 92% for RAPD and 85% for ISSR primers. The average of Polymorphism Information Content (PIC), Marker Index (MI), Nei’s gene diversity (H) and Shannon’s diversity index (I) in ISSR markers are higher than those in RAPD markers. Based on banding patterns and gene diversities values, we observed that the ISSR-PCR provides clearer data and is more successful in genetic diversity analyses than RAPD-PCR. Dendrograms generated from UPGMA (Unweighted Pair Group Method with Arithmetic Mean) cluster analyses for RAPD and ISSR markers were related to the geographic origin.

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.

Aflatoxin-producing fungi associated with pre-harvest maize contamination in Uganda

Maize is an important staple crop for the majority of the population in Uganda. However, in tropical and subtropical climates, maize is frequently contaminated with aflatoxins, a group of cancer-causing and immuno-suppressive mycotoxins produced by Aspergillus section Flavi fungi. In Uganda, there is limited knowledge about the causal agents of aflatoxin contamination. The current study determined both the aflatoxin levels in pre-harvest maize across Uganda and the structures of communities of aflatoxin-producing fungi associated with the maize. A total of 256 pre-harvest maize samples were collected from 23 major maize-growing districts in eight agro-ecological zones (AEZ). Maize aflatoxin content ranged from 0 to 3760 ng/g although only around 5% for Ugandan thresholds. For EU it is about 16% of the samples contained aflatoxin concentrations above tolerance thresholds. A total of 3105 Aspergillus section Flavi isolates were recovered and these were dominated by the A. flavus L morphotype (89.4%). Densities of aflatoxin-producing fungi were negatively correlated with elevation. Farming systems and climatic conditions of the AEZ are thought to have influenced communities' structure composition. Fungi from different AEZ varied significantly in aflatoxin-producing abilities and several atoxigenic genotypes were identified. The extremely high aflatoxin concentrations detected in some of the studied regions indicate that management strategies should be urgently designed for use at the pre-harvest stage. Atoxigenic genotypes detected across Uganda could serve as aflatoxin biocontrol agents to reduce crop contamination from fields conditions and throughout the maize value chain.

Aspergillus section Flavi diversity and the role of A. novoparasiticus in aflatoxin contamination in the sugarcane production chain

The presence of Aspergillus section Flavi and aflatoxins in sugarcane as well as in by-products, such as molasses, sugar, yeast cream and dried yeast, collected from different fields and processing plants in São Paulo state, were investigated throughout the sugarcane production chain. A total of 246 samples was collected and analyzed and 226 isolates of Aspergillus section Flavi were isolated. Aspergillus section Flavi strains were found in sugarcane juice, milled sugarcane, stalk, soil and dried yeast samples. Among the isolates of Aspergillus section Flavi submitted to polyphasic identification (n = 57), Aspergillus novoparasiticus and Aspergillus arachidicola were predominantly found. A significant proportion of the isolates (84.5%) were found to have morphological and physiological characteristics of A. novoparasiticus. Most samples, with the exception of sugar, showed some aflatoxin contamination. The highest level was in dried yeast with an average of 2.55 μg/kg and maximum value of 10.19 μg/kg. This is the first report of contamination of sugarcane by A. novoparasiticus.

Occurrence of Toxigenic Fungi and Aflatoxin Potential of Aspergillus spp. Strains Associated with Subsistence Farmed Crops in Haiti

Subsistence farming and poor storage facilities favor toxigenic fungal contamination and mycotoxin accumulation in staple foods from tropical countries such as Haiti. The present preliminary study was designed to evaluate the occurrence of toxigenic fungi in Haitian foodstuffs to define the mycotoxin risk associated with Haitian crops. The objectives of this research were to determine the distribution of toxigenic fungi in the Haitian crops maize, moringa, and peanut seeds and to screen Aspergillus section Flavi (ASF) isolates for production of aflatoxins B1 and G1 in vitro. Maize, moringa, and peanut samples were contaminated by potential toxigenic fungal taxa, mainly ASF and Fusarium spp. The isolation frequency of Aspergillus spp. and Fusarium spp. was influenced by locality and thus by farming systems, storage systems, and weather conditions. Particularly for ASF in peanut and maize samples, isolation frequencies were directly related to the growing season length. The present study represents the first report of contamination by toxigenic fungi and aflatoxin in moringa seeds, posing concerns about the safety of these seeds, which people in Haiti commonly consume. Most (80%) of the Haitian ASF strains were capable of producing aflatoxins, indicating that Haitian conditions clearly favor the colonization of toxigenic ASF strains over atoxigenic strains. ASF strains producing both aflatoxins B1 and G1 were found. Understanding the distribution of toxigenic ASF in Haitian crops and foodstuffs is important for determining accurate toxicological risks because the toxic profile of ASF is species specific. The occurrence of toxigenic fungi and the profiles of the ASF found in various crops highlight the need to prevent formation of aflatoxins in Haitian crops. This study provides relevant preliminary baseline data for guiding the development of legislation regulating the quality and safety of crops in this low-income country.

Study of the genetic diversity of the aflatoxin biosynthesis cluster in Aspergillus section Flavi using insertion/deletion markers in peanut seeds from Georgia, USA

ABSTRACTAflatoxins are among the most powerful carcinogens in nature. The major aflatoxin-producing fungi are Aspergillus flavus and A. parasiticus. Numerous crops, including peanut, are susceptible to aflatoxin contamination by these fungi. There has been an increased use of RNA interference (RNAi) technology to control phytopathogenic fungi in recent years. In order to develop molecular tools targeting specific genes of these fungi for the control of aflatoxins, it is necessary to obtain their genome sequences. Although high-throughput sequencing is readily available, it is still impractical to sequence the genome of every isolate. Thus, in this work, the authors proposed a workflow that allowed prescreening of 238 Aspergillus section Flavi isolates from peanut seeds from Georgia, USA. The aflatoxin biosynthesis cluster (ABC) of the isolates was fingerprinted at 25 InDel (insertion/deletion) loci using capillary electrophoresis. All isolates were tested for aflatoxins using ultra-high-performance liquid chromatography. The neighbor-joining, three-dimension (3D) principal coordinate, and Structure analyses revealed that the Aspergillus isolates sampled consisted of three main groups determined by their capability to produce aflatoxins. Group I comprised 10 non-aflatoxigenic A. flavus; Group II included A. parasiticus; and Group III included mostly aflatoxigenic A. flavus and the three non-aflatoxigenic A. caelatus. Whole genomes of 10 representative isolates from different groups were sequenced. Although InDels in Aspergillus have been used by other research groups, this is the first time that the cluster analysis resulting from fingerprinting was followed by whole-genome sequencing of representative isolates. In our study, cluster analysis of ABC sequences validated the results obtained with fingerprinting. This shows that InDels used here can predict similarities at the genome level. Our results also revealed a relationship between groups and their capability to produce aflatoxins. The database generated of Aspergillus spp. can be used to select target genes and assess the effectiveness of RNAi technology to reduce aflatoxin contamination in peanut.

FUNGAL CONTAMINATION AND MYCOTOXIN PRODUCTION BY ASPERGILLUS SPP. IN NUTS AND SESAME SEEDS

This work reports the occurrence of the fungal flora and evaluates the mycotoxigenic potential of Aspergillus genera in 63 samples of oil seeds and nuts (almonds, pistachio and sesame seeds). Fungal isolation and identification revealed the presence of 5 genera (Aspergillus, Penicillium, Cladosporium, Mucor and yeasts) with the predominance of Aspergillus section Nigri. A number of 138 strains of Aspergillus section Nigri and 91 of Aspergillus section Flavi were isolated and tested for their ability to produce ochratoxin A (OTA) and Aflatoxin, respectively. The detection of Aflatoxins and OTA production was carried out using thin-layer chromatography (TLC). Our results showed that 93.93% of Aspergillus carbonarius were able to produce OTA, but none of the Aspergillus niger aggregate was found to be an OTA-producer. Among the 91 Aspergillus section Flavi isolates, 88 were identified as Aspergillus flavus and 3 as Aspergillus parasiticus. All A. parasiticus were strong AFB and AFG producers. A percentage of 30.3% of A. flavus isolates produced AFB1, with levels ranging from 0.69 to 44.28 μg.g−1. The frequency of aflatoxigenic A. flavus strains was higher in pistachios (46.3%) than in almonds (30.3%) or sesame seeds (23.52%). Cyclopiazonic acid (CPA) and sclerotia production were carried out on CYA medium. All aflatoxigenic A. flavus strains produced CPA, whereas 29.2% produced sclerotia (L-type).

Mycobiota and Incidence of Toxigenic Fungi in Dried Fruits from Duhok Markets, North Iraq

Thirtysamples from each of four dried fruits (apricot,fig,plum and raisins) collectedfrom local shops at Duhok governorate were surveyed for their contaminationwith fungi. Thirty eight fungalspecies belonged to 13 genera in addition to yeasts were isolated andidentified. The highest diversity of fungi were detected from raisins (35species), followed by 27 species isolated from plum, 26 species from figs and18 species on apricot. Eleven species were found common on the four types ofdried fruits. These include Alternaria alternata, Aspergillus carbonarius,A. flavus, A.fumigatus, A.niger,A.parasiticus, Penicillium citrinum, P.expansum,Cladosporium cladosporoides, Emericella nidulans and Eurotium amstelodami.Aflatoxigenic potentials of selected isolates of Aspergillussection Flavi and ochratoxigenic potential of selected isolates from Aspergillussection Nigri were detected by ELISA technique. Aflatoxin was found atlevels from 79.4 to 356ppb whereas, ochratoxin A at levels from 60-106ppb.

Toxigenic molds in Tunisian and Egyptian sorghum for human consumption

The objective of this study was to characterize the mycoflora of sorghum grains commercialized in the Tunisian retail market and to identify aflatoxins (AFs), ochratoxin A (OTA) and zearalenone (ZEA) producing species. Sixty four samples of sorghum (37 samples of Tunisian sorghum and 27 samples of Egyptian sorghum) were analyzed. Dilution plating (CFU, colony forming units) was used for fungal enumeration. The isolation of mycobiota was carried out by plating of grains on PDA and malachite green medium. Aspergillus section Flavi and section Nigri and Fusarium isolates were sub-cultured in CYA to test their ability to produce AFs, OTA and ZEA, respectively. The selected Aspergillus section Flavi and section Nigri, Penicillium and Fusarium isolates were subjected to specific PCR assays using published species-specific primers. The results revealed the dominance of Fusarium (95.3%), followed by Aspergillus (87.2%) and Alternaria (81.2%) species. The fungal counts ranged from 100 to 1.3·104 CFU/g for Tunisian sorghum and from 100 to 5.7·103 CFU/g for Egyptian sorghum. Among Aspergillus section Flavi isolates identified by molecular biology, Aspergillus flavus was the most dominant (90.1%) while Aspergillus parasiticus represent 9.9% only. About Aspergillus section Nigri, results showed the dominance of Aspergillus niger aggregate species, including Aspergillus niger, Aspergillus tubingensis and other species. Among Fusarium species, Fusarium incarnatum was the most dominant in both Tunisian and Egyptian sorghum. Penicillium citrinum was the dominant Penicillium species in the studied samples. More than 890 isolates belonging to the genus Aspergillus and Fusarium were tested in order to test their capacity to produce AFs, OTA and ZEA. The percentage of mycotoxin producing isolates in Aspergillus section Flavi, A. section Nigri, and Fusarium was 30.0%, 4.6% and 11.1%, respectively.

Aflatoxin, cyclopiazonic acid and β-nitropropionic acid production byAspergillussectionFlavifrom dried figs grown in Turkey

Aspergillus section Flavi isolates from dried figs were screened for aflatoxins, cyclopiazonic acid (CPA) and β-nitropropionic acid (BNP) production on yeast extract sucrose, czapek yeast extract and potato dextrose agar media. According to molecular identification, Aspergillus flavus (n=51) was dominant followed by Aspergillus parasiticus (n=5) and Aspergillus tamarii (n=1). All isolates were toxigenic producing at least one of the mycotoxins. 98% of the A. flavus strains produced aflatoxins (0.3-1,620 ng/g) and CPA (0.07-398.2 μg/g), with 31% of them also producing BNP (7.9-1,571.2 ng/g). All isolates of A. parasiticus were aflatoxins producers (106.4-1,255 ng/g), while 2 of them (40%) also produced CPA at a very low level (0.05-0.06 μg/g). Regarding toxigenic profiles of A. flavus and A. parasiticus, 6 and 3 chemotypes were classified, respectively. A. flavus isolates producing both B type of aflatoxins and CPA, comprised the most represented chemotype followed by the chemotype consisting of aflatoxin B1, CPA and BNP producers. Regarding A. parasiticus chemotypes, two can be considered atypical being producer of only the B type of aflatoxins and being producer of the B, G type of aflatoxins along with CPA. To the best of our knowledge, an A. flavus isolate producing CPA and BNP but not aflatoxin has been found for the first time.

Distribution of aflatoxigenic Aspergillus section Flavi in commercial poultry feed in Nigeria

The distribution and aflatoxigenicity of Aspergillus section Flavi isolates in 58 commercial poultry feed samples obtained from 17 states in five agro-ecological zones (AEZs) in Nigeria were determined in order to assess the safety of the feeds with respect to aflatoxin-producing fungi. Correlation was also performed for incidence of species, aflatoxin-producing ability of isolates in vitro, and aflatoxin (AFB1) concentrations in the feed. A total of 1006 Aspergillus section Flavi isolates were obtained from 87.9% of the feed samples and identified as Aspergillus flavus, unnamed taxon SBG, Aspergillus parasiticus and Aspergillus tamarii. A. flavus was the most prevalent (91.8%) of the isolates obtained from the feed in the AEZs while A. parasiticus had the lowest incidence (0.1%) and was isolated only from a layer mash sample collected from the DS zone. About 29% of the Aspergillus isolates produced aflatoxins in maize grains at concentrations up to 440,500μg/kg B and 341,000μg/kgG aflatoxins. The incidence of toxigenic isolates was highest (44.4%) in chick mash and lowest (19.9%) in grower mash. The population of A. flavus in the feed had positive (r=0.50) but non significant (p>0.05) correlations with proportion of toxigenic isolates obtained from the feed while SBG had significant (p<0.001) positive (r=0.99) influence on AFB1 concentrations in the feed. Poultry feed in Nigerian markets are therefore highly contaminated with aflatoxigenic Aspergillus species and consequently, aflatoxins. This is a potential threat to the poultry industry and requires urgent intervention.