Aflatoxin profiles of Aspergillus flavus isolates in Sudanese fungal rhinosinusitis.

Aspergillus flavus is a commonly encountered pathogen responsible for fungal rhinosinusitis (FRS) in arid regions. The species is known to produce aflatoxins, posing a significant risk to human health. This study aimed to investigate the aflatoxin profiles of A. flavus isolates causing FRS in Sudan. A total of 93 clinical and 34 environmental A. flavus isolates were studied. Aflatoxin profiles were evaluated by phenotypic (thin-layer and high-performance chromatography) and genotypic methods at various temperatures and substrates. Gene expression of aflD and aflR was also analyzed. A total of 42/93 (45%) isolates were positive for aflatoxin B1 and AFB2 by HPLC. When the incubation temperature changed from 28°C to 36°C, the number of positive isolates decreased to 41% (38/93). Genetic analysis revealed that 85% (79/93) of clinical isolates possessed all seven aflatoxin biosynthesis-associated genes, while 27% (14/51) of non-producing isolates lacked specific genes (aflD/aflR/aflS). Mutations were observed in aflS and aflR genes across both aflatoxin-producers and non-producers. Gene expression of aflD and aflR showed the highest expression between the 4th and 6th days of incubation on the Sabouraud medium and on the 9th day of incubation on the RPMI (Roswell Park Memorial Institute) medium. Aspergillus flavus clinical isolates demonstrated aflatoxigenic capabilities, influenced by incubation temperature and substrate. Dynamic aflD and aflR gene expression patterns over time enriched our understanding of aflatoxin production regulation. The overall findings underscored the health risks of Sudanese patients infected by this species, emphasizing the importance of monitoring aflatoxin exposure.

Aspergillus flavus, mainly causing fungal rhinosinusitis in Sudan, poses health risks due to aflatoxin production. This study revealed diverse levels of aflatoxin and gene expression of clinical isolates by pheno- and genotypic methods, emphasizing the need for vigilant monitoring in the region.

New Insights of Transcriptional Regulator AflR in Aspergillus flavus Physiology.

Aspergillus flavus aflR, a gene encoding a Zn(II)2Cys6 DNA-binding domain, is an important transcriptional regulator of the aflatoxin biosynthesis gene cluster. Our previous results of Gene ontology (GO) analysis for the binding sites of AflR in A. flavus suggest that AflR may play an integrative regulatory role. In this study the ΔaflR and overexpression (OE) strains based on the well-established double-crossover recombinational technique were constructed to investigate the integrative function of the aflR gene in A. flavus. The disruption of aflR severely affected the aflatoxin biosynthetic pathway, resulting in a significant decrease in aflatoxin production. The aflatoxin B1 (AFB1) of the ΔaflR strain was 180 ng/mL and aflatoxin B2 (AFB2) was 2.95 ng/mL on YES medium for 5 days, which was 1/1,000 of that produced by the wild-type strain (WT). In addition, the ΔaflR strain produced relatively sparse conidia and a very small number of sclerotia. On the seventh day, the sclerotia yield on each plate of the WT and OE strains exceeded 1,000, while the sclerotial formation of the ΔaflR strain was not detected until 14 days. However, the biosynthesis of cyclopiazonic acid (CPA) was not affected by aflR gene disruption. Transcriptomic analysis of the ΔaflR strain grown on potato dextrose agar (PDA) plates at 0 h, 24 h, and 72 h showed that expression of clustering genes involved in the biosynthesis of aflatoxin was significantly downregulated. Meanwhile, the ΔaflR strain compared with the WT strain showed significant expression differences in genes involved in spore germination, sclerotial development, and carbohydrate metabolism compared to the WT. The results demonstrated that the A. flavus aflR gene also played a positive role in the fungal growth and development in addition to aflatoxin biosynthesis. IMPORTANCE Past studies of the A. flavus aflR gene and its orthologues in related Aspergillus species were solely focused on their roles in secondary metabolism. In this study, we used the ΔaflR and OE strains to demonstrate the role of aflR in growth and development of A. flavus. For the first time, we confirmed that the ΔaflR strain also was defective in production of conidia and sclerotia, asexual propagules of A. flavus. Our transcriptomic analysis further showed that genes involved in spore germination, sclerotial development, aflatoxin biosynssssthesis, and carbohydrate metabolism exhibited significant differences in the ΔaflR strain compared with the WT strain. Our study indicates that AflR not only plays an important role in regulating aflatoxin synthesis but also in playing a positive role in the conidial formation and sclerotial development in A. flavus. This study reveals the critical and positive role of the aflR gene in fungal growth and development, and provides a theoretical basis for the genetic studies of other aspergilli.

Microevolution in the pansecondary metabolome of Aspergillus flavus and its potential macroevolutionary implications for filamentous fungi.

Fungi produce a wealth of pharmacologically bioactive secondary metabolites (SMs) from biosynthetic gene clusters (BGCs). It is common practice for drug discovery efforts to treat species' secondary metabolomes as being well represented by a single or a small number of representative genomes. However, this approach misses the possibility that intraspecific population dynamics, such as adaptation to environmental conditions or local microbiomes, may harbor novel BGCs that contribute to the overall niche breadth of species. Using 94 isolates of Aspergillus flavus, a cosmopolitan model fungus, sampled from seven states in the United States, we dereplicate 7,821 BGCs into 92 unique BGCs. We find that more than 25% of pangenomic BGCs show population-specific patterns of presence/absence or protein divergence. Population-specific BGCs make up most of the accessory-genome BGCs, suggesting that different ecological forces that maintain accessory genomes may be partially mediated by population-specific differences in secondary metabolism. We use ultra-high-performance high-resolution mass spectrometry to confirm that these genetic differences in BGCs also result in chemotypic differences in SM production in different populations, which could mediate ecological interactions and be acted on by selection. Thus, our results suggest a paradigm shift that previously unrealized population-level reservoirs of SM diversity may be of significant evolutionary, ecological, and pharmacological importance. Last, we find that several population-specific BGCs from A. flavus are present in Aspergillus parasiticus and Aspergillus minisclerotigenes and discuss how the microevolutionary patterns we uncover inform macroevolutionary inferences and help to align fungal secondary metabolism with existing evolutionary theory.

Effects of chlorpyrifos on growth and aflatoxin B1 production by Aspergillus section Flavi strains on maize-based medium and maize grains.

Chlorpyrifos is one of the most used insecticides in agro-ecosystems and is repeatedly applied due to the increase in pest resistance, which leads to environmental accumulation. The aim of this work was to evaluate the effect of chlorpyrifos on growth and aflatoxin B1 (AFB1) production by four Aspergillus section Flavi strains, under different water conditions-aW (0.93, 0.95 and 0.98)-on maize-based medium (MMEA) and maize grains supplied with 0.06 to 1.4 mmol/L of chlorpyrifos. MMEA plates were incubated at 18, 28, and 37 °C and plates with maize grains at 25 °C during 21 days. Chlorpyrifos stimulated the growth and AFB1 production of non-target organisms, such as Aspergillus section Flavi strains, both at low (0.06 mmol/L) and at high concentrations (1.4 mmol/L) on MMEA and maize grains. Stimulation occurred over a wide range of temperature and aw conditions. The toxin concentration produced by the two strains on MMEA at 18 °C increased when the concentration of chlorpyrifos also increased, being most significant at 0.6 mmol/L. In conclusion, the presence of chlorpyrifos should be considered as a factor, together with environmental conditions, for the development of effective production practices of maize grains, in order to avoid fungal growth and AFB1 production, to prevent both economic losses and risks to human and animal health.

Crystal structure of a S-adenosyl-L-methionine-dependent O-methyltransferase-like enzyme from Aspergillus flavus.

S-adenosyl-L-methionine (SAM)-dependent methyltransferases (MTases) are widely distributed among almost all organisms and often characterized with conserved Rossmann fold, TIM barrel, and D×G×G×G motif. However, some MTases show no methyltransferase activity. In the present study, the crystal structure of LepI, one MTase-like enzyme isolated from A. flavus that catalyzes pericyclic reactions, was investigated to determine its structure-function relationship. The overall structure of LepI in complex with the SAM mimic S-adenosyl-L-homocysteine (SAH) (PDB ID: 6IV7) indicated that LepI is a tetramer in solution. The residues His133, Arg197, Arg295, and Asp296 located near the active site can form hydrogen bonds with the substrate, thus participating in catalytic reactions. The binding of SAH in LepI is almost identical to that in other resolved MTases; however, the location of catalytic residues differs significantly. Phylogenetic trials suggest that LepI proteins share a common ancestor in plants and algae, which may explain the conserved SAM-binding site. However, the accelerated evolution of A. flavus has introduced both functional and structural changes in LepI. More importantly, the residue Arg295, which is unique to LepI, might be a key determinant for the altered enzymatic behavior. Collectively, the differences in the composition of catalytic residues, as well as the unique tetrameric form of LepI, define its unique enzymatic behavior. The present work provides an additional understanding of the structure-function relationship of MTases and MTase-like enzymes.

Genetic diversity of aflatoxin-producing Aspergillus flavus isolated from selected groundnut growing agro-ecological zones of Uganda.

BACKGROUND: Groundnut pre- and post-harvest contamination is commonly caused by fungi from the Genus Aspergillus. Aspergillus flavus is the most important of these fungi. It belongs to section Flavi; a group consisting of aflatoxigenic (A. flavus, A. parasiticus and A. nomius) and non-aflatoxigenic (A. oryzae, A. sojae and A. tamarii) fungi. Aflatoxins are food-borne toxic secondary metabolites of Aspergillus species associated with severe hepatic carcinoma and children stuntedness. Despite the well-known public health significance of aflatoxicosis, there is a paucity of information about the prevalence, genetic diversity and population structure of A. flavus in different groundnut growing agro-ecological zones of Uganda. This cross-sectional study was therefore conducted to fill this knowledge gap. RESULTS: The overall pre- and post-harvest groundnut contamination rates with A. flavus were 30.0 and 39.2% respectively. Pre- and post-harvest groundnut contamination rates with A. flavus across AEZs were; 2.5 and 50.0%; (West Nile), 55.0 and 35.0% (Lake Kyoga Basin) and 32.5 and 32.5% (Lake Victoria Basin) respectively. There was no significant difference (χ2 = 2, p = 0.157) in overall pre- and post-harvest groundnut contamination rates with A. flavus and similarly no significant difference (χ2 = 6, p = 0.199) was observed in the pre- and post-harvest contamination of groundnut with A. flavus across the three AEZs. The LKB had the highest incidence of aflatoxin-producing Aspergillus isolates while WN had no single Aspergillus isolate with aflatoxin-producing potential. Aspergillus isolates from the pre-harvest groundnut samples had insignificantly higher incidence of aflatoxin production (χ2 = 2.667, p = 0.264) than those from the post-harvest groundnut samples. Overall, A. flavus isolates exhibited moderate level (92%, p = 0.02) of genetic diversity across the three AEZs and low level (8%, p = 0.05) of genetic diversity within the individual AEZs. There was a weak positive correlation (r = 0.1241, p = 0.045) between genetic distance and geographic distance among A. flavus populations in the LKB, suggesting that genetic differentiation in the LKB population might be associated to geographic distance. A very weak positive correlation existed between genetic variation and geographic location in the entire study area (r = 0.01, p = 0.471), LVB farming system (r = 0.0141, p = 0.412) and WN farming system (r = 0.02, p = 0.478). Hierarchical clustering using the unweighted pair group method with arithmetic means (UPGMA) revealed two main clusters of genetically similar A. flavus isolates. CONCLUSIONS: These findings provide evidence that genetic differentiation in A. flavus populations is independent of geographic distance. This information can be valuable in the development of a suitable biocontrol management strategy of aflatoxin-producing A. flavus.

The Frequency of Sex: Population Genomics Reveals Differences in Recombination and Population Structure of the Aflatoxin-Producing Fungus Aspergillus flavus.

The apparent rarity of sex in many fungal species has raised questions about how much sex is needed to purge deleterious mutations and how differences in frequency of sex impact fungal evolution. We sought to determine how differences in the extent of recombination between populations of Aspergillus flavus impact the evolution of genes associated with the synthesis of aflatoxin, a notoriously potent carcinogen. We sequenced the genomes of, and quantified aflatoxin production in, 94 isolates of A. flavus sampled from seven states in eastern and central latitudinal transects of the United States. The overall population is subdivided into three genetically differentiated populations (A, B, and C) that differ greatly in their extent of recombination, diversity, and aflatoxin-producing ability. Estimates of the number of recombination events and linkage disequilibrium decay suggest relatively frequent sex only in population A. Population B is sympatric with population A but produces significantly less aflatoxin and is the only population where the inability of nonaflatoxigenic isolates to produce aflatoxin was explained by multiple gene deletions. Population expansion evident in population B suggests a recent introduction or range expansion. Population C is largely nonaflatoxigenic and restricted mainly to northern sampling locations through restricted migration and/or selection. Despite differences in the number and type of mutations in the aflatoxin gene cluster, codon optimization and site frequency differences in synonymous and nonsynonymous mutations suggest that low levels of recombination in some A. flavus populations are sufficient to purge deleterious mutations.IMPORTANCE Differences in the relative frequencies of sexual and asexual reproduction have profound implications for the accumulation of deleterious mutations (Muller's ratchet), but little is known about how these differences impact the evolution of ecologically important phenotypes. Aspergillus flavus is the main producer of aflatoxin, a notoriously potent carcinogen that often contaminates food. We investigated if differences in the levels of production of aflatoxin by A. flavus could be explained by the accumulation of deleterious mutations due to a lack of recombination. Despite differences in the extent of recombination, variation in aflatoxin production is better explained by the demography and history of specific populations and may suggest important differences in the ecological roles of aflatoxin among populations. Furthermore, the association of aflatoxin production and populations provides a means of predicting the risk of aflatoxin contamination by determining the frequencies of isolates from low- and high-production populations.

Molecular profile of non-aflatoxigenic phenotype in native strains of Aspergillus flavus.

Aflatoxins are the most common mycotoxin contaminant reported in food and feed. Aflatoxin B1, the most toxic among different aflatoxins, is known to cause hepatocellular carcinoma in animals. Aspergillus flavus and A. parasiticus are the main producers of aflatoxins and are widely distributed in tropical countries. Even though several robust strategies have been in use to control aflatoxin contamination, the control at the pre-harvest level is primitive and incompetent. Therefore, the aim of the study was to isolate and identify the non-aflatoxigenic A. flavus and to delineate the molecular mechanism for the loss of aflatoxin production by the non-aflatoxigenic isolates. Eighteen non-aflatoxigenic strains were isolated from various biological sources using cultural and analytical methods. Among the 18 isolates, 8 isolates produced sclerotia and 17 isolates had type I deletion in norB-cypA region. The isolates were confirmed as A. flavus using gene-specific PCR and sequencing of the ITS region. Later, aflatoxin gene-specific PCR revealed that the defect in one or more genes has led to non-aflatoxigenic phenotype. The strain R9 had maximum defect, and genes avnA and verB had the highest frequency of defect among the non-aflatoxigenic strains. Further, qRT-PCR confirmed that the non-aflatoxigenic strains had high frequency of defect or downregulation in the late pathway genes compared to early pathway genes. Thus, these non-aflatoxigenic strains can be the potential candidates for an effective and proficient strategy for the control of pre-harvest aflatoxin contamination.

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.

Detection of virulence factors and antifungal susceptibility of human and avian Aspergillusflavus isolates.

Aspergillusflavus is the second leading cause of invasive and non-invasive aspergillosis. Secretion of hydrolytic enzymes is considered as a virulence factor in this species. Our work aimed to study in vitro production of some virulence factors, to evaluate the biofilm production against human and avian A. flavus isolates and to investigate the antifungal susceptibility agents. Hydrolytic enzymes, biofilm production and molecular typing were studied for 62 human and 36 avian A. flavus isolates by specific solid media and six microsatellite markers. The susceptibility to antifungal agents was evaluated for 37 human isolates. All human and avian A. flavus isolates showed positive activities of extracellular hydrolase: phospholipase, protease and hemolysin. A positive elastase activity was seen in 64.51% of human A. flavus isolates and 86.1% of avian A. flavus isolates. All A. flavus in these two populations formed biofilms. Statistical significant difference was observed for the mean phospholipase activities (P=0.025) and biofilm quantification (P=0.0001) between human and avian A. flavus isolates. The in vitro susceptibility results showed a resistance in 83.7%, 81.08% and 16.21% of A. flavus isolates respectively to amphotericin B, itraconazole and posaconazole. No association was noted between all virulence factors and the genotypes of human and avian isolates. Our study allowed us to show that human strains have a higher production of extracellular hydrolases and biofilm then avian strains. These virulence factors appear to act synergistically to contribute to the virulence of A. flavus strains. Moreover, significant correlation between virulence patterns and antifungal susceptibility profiles was observed.

Discrimination of Aspergillus flavus from Aspergillus oryzae by matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry.

BACKGROUND: Aspergillus flavus is a major cause of severe non-invasive fungal infections in the Middle Eastern countries. However, it is difficult to distinguish A flavus from A oryzae. OBJECTIVES: To assess the potential of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) in discriminating between A flavus and A oryzae and compare it with ß-tubulin gene sequencing. METHODS: We used the Bruker Daltonik MALDI-TOF MS system to analyse 200 clinical and environmental A flavus isolates and one A pseudonomius and one A alliaceus (Aspergillus section Flavi) isolate a priori identified as such by sequencing of the ß-tubulin gene. RESULTS: All 200 A flavus isolates were identified at the genus level and 176 (88%) at the species levels by MALDI-TOF MS based on the spectral log-scores (≥2.0 and 1.7-1.99, respectively); among them, only 18 (10.2%) were confirmed as A flavus, whereas 35 (19.9%) were identified as A oryzae and 123 (69.9%) as A flavus/A oryzae. Aspergillus pseudonomius and A alliaceus were misidentified as A flavus and A parasiticus with log-score values of 1.39 and 1.09, respectively. CONCLUSIONS: The results indicate that the commercially available Bruker Daltonik MALDI-TOF MS score database cannot separate A flavus and A oryzae species. We also showed that establishment of an in-house library is a useful tool to discriminate closely related Aspergillus species, including A flavus and A oryzae.

Draft genome sequence of Aspergillus flavus isolate TERIBR1, a highly tolerant fungus to chromium stress.

OBJECTIVES: Aspergillus flavus isolate TERIBR1 was isolated from tannery sludge highly contaminated with chromium. During characterization process, it exhibited capability to adapt and grow in fungal growth media amended with chromium concentration as high as 250 mg/l. In order to understand the genetic underpinnings of the chromium tolerance trait, whole genome sequencing of the TERIBR1 genome was carried out. Information from the current genome will facilitate an understanding of the mechanisms underlying fungal adaptation to heavy metal stress and also heavy metal bioremediation. DATA DESCRIPTION: Here, we report the draft genome sequence along with the assembly and annotation methods used for genome sequence of the A. flavus isolate TERIBR1. The draft genome assembly size is estimated at 37.7 Mb coding for 13,587 genes and has high similarity to the reference genome of A. flavus strain NRRL3357.

Morphologic, molecular and metabolic characterization of Aspergillus section Flavi in spices marketed in Lebanon.

Spices are used extensively in Lebanon not only to flavour foods but also for their medicinal properties. To date, no data are available regarding the nature of the toxigenic fungal species that may contaminate these products at the marketing stage in this country. Eighty samples corresponding to 14 different types of spices were collected throughout Lebanon to characterize the Aspergillus section Flavi contaminating spices marketed in Lebanon and the toxigenic potential of these fungal species. Most fungal genera and species were identified as belonging to Aspergillus section Flavi. Aspergillus flavus was the most frequent species, representing almost 80% of the isolates. Although identified as A. flavus by molecular analysis, some strains displayed atypical morphological features. Seven strains of A. tamarii and one A. minisclerotigenes were also isolated. Analyses of toxigenic potential demonstrated that almost 80% of strains were able to produce mycotoxins, 47% produced aflatoxins, and 72% produced cyclopiazonic acid, alone or in combination with aflatoxins.

Characterization of Aspergilli from dried red chilies (Capsicum spp.): Insights into the etiology of aflatoxin contamination.

Aflatoxins are toxic carcinogens produced by several species of Aspergillus section Flavi, with some aflatoxin producers associated with specific crops. Red chilies (Capsicum spp.) are grown in warm regions that also favor aflatoxin-producers. Aflatoxins in red chilies may result in serious health concerns and severe economic losses. The current study sought to gain insight on causal agents of aflatoxin contamination in red chilies. Naturally contaminated chilies from markets in Nigeria (n = 55) and the United States (US) (n = 169) were examined. The A. flavus L strain was the predominant member of Aspergillus section Flavi (84%) in chilies. Highly toxigenic fungi with S strain morphology were also detected in chilies from both countries (11%), followed by A. tamarii (4.6%) and A. parasiticus (0.4%). Fungi with L morphology produced significantly lower quantities of aflatoxins (mean = 43 µg g-1) compared to S morphology fungi (mean = 667 µg g-1; p < 0.01) in liquid fermentation. Eighty-one percent of S morphology fungi from chilies in US markets produced only B aflatoxins, whereas 20%, all imported from Nigeria, produced both B and G aflatoxins; all S morphology fungi from Nigerian chilies produced both B and G aflatoxins. Multi-gene phylogenetic analyses of partial gene sequences for nitrate reductase (niaD, 2.1 kb) and the aflatoxin pathway transcription factor (aflR, 1.9 kb) resolved Aspergilli recovered from chilies into five highly supported distinct clades: 1) A. parasiticus; 2) A. flavus with either L or S morphology; 3) A. minisclerotigenes; 4) A. aflatoxiformans, and 5) a new lineage. Aspergillus aflatoxiformans and the new lineage produced the highest concentrations of total aflatoxins in chilies, whereas A. flavus L strains produced the least. The results suggest etiology of aflatoxin contamination of chili is complex and may vary with region. Knowledge of causal agents of aflatoxin contamination of chilies will be helpful in developing mitigation strategies to prevent human exposure.

Whole genome comparison of Aspergillus flavus L-morphotype strain NRRL 3357 (type) and S-morphotype strain AF70.

Aspergillus flavus is a saprophytic fungus that infects corn, peanuts, tree nuts and other agriculturally important crops. Once the crop is infected the fungus has the potential to secrete one or more mycotoxins, the most carcinogenic of which is aflatoxin. Aflatoxin contaminated crops are deemed unfit for human or animal consumption, which results in both food and economic losses. Within A. flavus, two morphotypes exist: the S strains (small sclerotia) and L strains (large sclerotia). Significant morphological and physiological differences exist between the two morphotypes. For example, the S-morphotypes produces sclerotia that are smaller (< 400 µm), greater in quantity, and contain higher concentrations of aflatoxin than the L-morphotypes (>400 µm). The morphotypes also differ in pigmentation, pH homeostasis in culture and the number of spores produced. Here we report the first full genome sequence of an A. flavus S morphotype, strain AF70. We provide a comprehensive comparison of the A. flavus S-morphotype genome sequence with a previously sequenced genome of an L-morphotype strain (NRRL 3357), including an in-depth analysis of secondary metabolic clusters and the identification SNPs within their aflatoxin gene clusters.

Characterization and competitive ability of non-aflatoxigenic Aspergillus flavus isolated from the maize agro-ecosystem in Argentina as potential aflatoxin biocontrol agents.

Aspergillus flavus is an opportunistic pathogen and may produce aflatoxins in maize, one of the most important crops in Argentina. A promising strategy to reduce aflatoxin accumulation is the biological control based on competitive exclusion. In order to select potential biocontrol agents among isolates from the maize growing region in Argentina, a total of 512 A. flavus strains were isolated from maize kernels and soil samples. Thirty-six per cent of the isolates from maize kernels did not produce detectable levels of aflatoxins, while 73% of the isolates from soil were characterized as non-aflatoxin producers. Forty percent and 49% of the isolates from maize kernels and soil samples, respectively, were not producers of cyclopiazonic acid (CPA). Sclerotia morphology was evaluated using Czapek Dox media. Eighty-six per cent of the isolates from maize kernels and 85% of the isolates from soil samples were L sclerotia morphotype (average diameter > 400 µm). The remaining isolates did not produce sclerotia. All isolates had MAT 1-1 idiomorph. The competitive ability of 9 non aflatoxigenic strains, 4 CPA(+) and 5 CPA(-), was evaluated in co-inoculations of maize kernels with an aflatoxigenic strain. All evaluated strains significantly (p < 0.05) reduced aflatoxin contamination in maize kernels. The aflatoxin B1 (AFB1) reduction ranged from 6 to 60%. The strain A. flavus ARG5/30 isolated from maize kernels would be a good candidate as a potential biocontrol agent to be used in maize, since it was characterized as neither aflatoxin nor CPA producer, morphotype L, MAT 1-1 idiomorph, and reduced AFB1 content in maize kernels by 59%. This study showed the competitive ability of potential aflatoxin biocontrol agents to be evaluated under field trials in a maize agro-ecosystem in Argentina.

Vegetative compatibility and phenotypic characterization as a means of determining genetic diversity of Aspergillus flavus isolates.

Toxigenic Aspergillus species produce mycotoxins that are carcinogenic, hepatotoxic and teratogenic immunosuppressing agents in both human and animals. Kenya frequently experiences outbreaks of aflatoxicosis with the worst occurring in 2010, which resulted in 215 deaths. We examined the possible reasons for these frequent aflatoxicosis outbreaks in Kenya by studying Aspergillus flavus diversity, phenotypes and mycotoxin profiles across various agricultural regions. Using diagonal transect random sampling, maize kernels were collected from Makueni, Homa Bay, Nandi, and Kisumu counties. Out of 37 isolates, nitrate non-utilizing auxotrophs complementation test revealed 20 vegetative compatibility groups. We designated these groups by the prefix "KVCG", where "K" represented Kenya and consequently assigned numbers 1-20 based on our findings. KVCG14 and KVCG15 had highest distribution frequency (n = 13; 10.8 %). The distribution of the L-, S- and S-/L-morphotypes across the regions were 57 % (n = 21); 7 % (n = 3) and 36 % (n = 13), respectively. Furthermore, a unique isolate (KSM015) was identified that had characteristics of S-morphotype, but produced both aflatoxins B and G. Coconut agar medium (CAM) assay, TLC and HPLC analyses confirmed the presence or absence of aflatoxins in selected toxigenic and atoxigenic isolates. Diversity index (H') analyses ranged from 0.11 (Nandi samples) to 0.32 (Kisumu samples). Heterokaryon compatibility ranged from 33 % (for the Makueni samples, n = 3) to 67 % (Nandi samples, n = 6). To our knowledge, this is the first reported findings for A. flavus diversity and distribution in Nandi, Homa Bay and Kisumu counties and may assist current and future researchers in the selection of biocontrol strategies to mitigate aflatoxin contamination as has been researched in Makueni and neighbouring counties.

Development of a droplet digital PCR assay for population analysis of aflatoxigenic and atoxigenic Aspergillus flavus mixtures in soil.

Aflatoxin B1 is a potent hepatotoxin and carcinogen that poses a serious safety hazard to both humans and animals. Aspergillus flavus is the most common aflatoxin-producing species on corn, cotton, peanuts, and tree nuts. Application of atoxigenic strains to compete against aflatoxigenic strains of A. flavus has emerged as one of the most practical strategies for ameliorating aflatoxin contamination in food. Genes directly involved in aflatoxin biosynthesis are clustered on an 82-kb region of the genome. Three atoxigenic strains (CA12, M34, and AF123) were each paired with each of four aflatoxigenic strains (CA28, CA42, CA90, and M52), inoculated into soil and incubated at 28 °C for 2 weeks and 1 month. TaqMan probes, omtA-FAM, and norA-HEX were designed for developing a droplet digital PCR (ddPCR) assay to analyze the soil population of mixtures of A. flavus strains. DNA was extracted from each soil sample and used for ddPCR assays. The data indicated that competition between atoxigenic and aflatoxigenic was strain dependent. Variation in competitive ability among different strains of A. flavus influenced the population reduction of the aflatoxigenic strain by the atoxigenic strain. Higher ratios of atoxigenic to aflatoxigenic strains increased soil population of atoxigenic strains. This is the first study to demonstrate the utility of ddPCR to quantify mixtures of both atoxigenic and aflatoxigenic A. flavus strains in soil and allows for rapid and accurate determination of population sizes of atoxigenic and aflatoxigenic strains. This method eliminates the need for isolation and identification of individual fungal isolates from experimental soil samples.

Optimal pcr primers for rapid and accurate detection of Aspergillus flavus isolates.

Aspergillus flavus is among the most devastating opportunistic pathogens of several food crops including rice, due to its high production of carcinogenic aflatoxins. The presence of these organisms in economically important rice strip farming is a serious food safety concern. Several polymerase chain reaction (PCR) primers have been designed to detect this species; however, a comparative assessment of their accuracy has not been conducted. This study aims to identify the optimal diagnostic PCR primers for the identification of A. flavus, among widely available primers. We isolated 122 A. flavus native isolates from randomly collected rice strips (N = 300). We identified 109 isolates to the genus level using universal fungal PCR primer pairs. Nine pairs of primers were examined for their PCR diagnostic specificity on the 109 isolates. FLA PCR was found to be the optimal PCR primer pair for specific identification of the native isolates, over aflP(1), aflM, aflA, aflD, aflP(3), aflP(2), and aflR. The PEP primer pair was found to be the most unsuitable for A. flavus identification. In conclusion, the present study indicates the powerful specificity of the FLA PCR primer over other commonly available diagnostic primers for accurate, rapid, and large-scale identification of A. flavus native isolates. This study provides the first simple, practical comparative guide to PCR-based screening of A. flavus infection in rice strips.

Probability models for growth and aflatoxin B1 production as affected by intraspecies variability in Aspergillus flavus.

The probability of growth and aflatoxin B1 (AFB1) production of 20 isolates of Aspergillus flavus were studied using a full factorial design with eight water activity levels (0.84-0.98 aw) and six temperature levels (15-40 °C). Binary data obtained from growth studies were modelled using linear logistic regression analysis as a function of temperature, water activity and time for each isolate. In parallel, AFB1 was extracted at different times from newly formed colonies (up to 20 mm in diameter). Although a total of 950 AFB1 values over time for all conditions studied were recorded, they were not considered to be enough to build probability models over time, and therefore, only models at 30 days were built. The confidence intervals of the regression coefficients of the probability of growth models showed some differences among the 20 growth models. Further, to assess the growth/no growth and AFB1/no- AFB1 production boundaries, 0.05 and 0.5 probabilities were plotted at 30 days for all of the isolates. The boundaries for growth and AFB1 showed that, in general, the conditions for growth were wider than those for AFB1 production. The probability of growth and AFB1 production seemed to be less variable among isolates than AFB1 accumulation. Apart from the AFB1 production probability models, using growth probability models for AFB1 probability predictions could be, although conservative, a suitable alternative. Predictive mycology should include a number of isolates to generate data to build predictive models and take into account the genetic diversity of the species and thus make predictions as similar as possible to real fungal food contamination.