Aflatoxin Contamination: An Overview on Health Issues, Detection and Management Strategies.

Aflatoxins (AFs) represent one of the main mycotoxins produced by Aspergillus flavus and Aspergillus parasiticus, with the most prevalent and lethal subtypes being AFB1, AFB2, AFG1, and AFG2. AFs are responsible for causing significant public health issues and economic concerns that affect consumers and farmers globally. Chronic exposure to AFs has been linked to liver cancer, oxidative stress, and fetal growth abnormalities among other health-related risks. Although there are various technologies, such as physical, chemical, and biological controls that have been employed to alleviate the toxic effects of AF, there is still no clearly elucidated universal method available to reduce AF levels in food and feed; the only mitigation is early detection of the toxin in the management of AF contamination. Numerous detection methods, including cultures, molecular techniques, immunochemical, electrochemical immunosensor, chromatographic, and spectroscopic means, are used to determine AF contamination in agricultural products. Recent research has shown that incorporating crops with higher resistance, such as sorghum, into animal feed can reduce the risk of AF contamination in milk and cheese. This review provides a current overview of the health-related risks of chronic dietary AF exposure, recent detection techniques, and management strategies to guide future researchers in developing better detection and management strategies for this toxin.

Intraspecific Growth and Aflatoxin Inhibition Responses to Atoxigenic Aspergillus flavus: Evidence of Secreted, Inhibitory Substances in Biocontrol.

The fungus Aspergillus flavus infects corn, peanut, and cottonseed, and contaminates seeds with acutely poisonous and carcinogenic aflatoxin. Aflatoxin contamination is a perennial threat in tropical and subtropical climates. Nonaflatoxin-producing isolates (atoxigenic) are deployed in fields to mitigate aflatoxin contamination. The biocontrol competitively excludes toxigenic A. flavus via direct replacement and thigmoregulated (touch) toxin inhibition mechanisms. To understand the broad-spectrum toxin inhibition, toxigenic isolates representing different mating types and sclerotia sizes were individually cocultured with different atoxigenic biocontrol isolates. To determine whether more inhibitory isolates had a competitive advantage to displace or touch inhibit toxigenic isolates, biomass accumulation rates were determined for each isolate. Finally, to determine whether atoxigenic isolates could inhibit aflatoxin production without touch, atoxigenic isolates were grown separated from a single toxigenic isolate by a membrane. Atoxigenic isolates 17, Af36, and K49 had superior abilities to inhibit toxin production. Small (<400 µm) sclerotial, Mat1-1 isolates were not as completely inhibited as others by most atoxigenic isolates. As expected for both direct replacement and touch inhibition, the fastest-growing atoxigenic isolates inhibited aflatoxin production the most, except for atoxigenic Af36 and K49. Aflatoxin production was inhibited when toxigenic and atoxigenic isolates were grown separately, especially by slow-growing atoxigenic Af36 and K49. Additionally, fungus-free filtrates from atoxigenic cultures inhibited aflatoxin production. Toxin production inhibition without direct contact revealed secretion of diffusible chemicals as an additional biocontrol mechanism. Biocontrol formulations should be improved by identifying isolates with broad-spectrum, high-inhibition capabilities and production of secreted inhibitory chemicals.

Genetic Responses and Aflatoxin Inhibition during Co-Culture of Aflatoxigenic and Non-Aflatoxigenic Aspergillus flavus.

Aflatoxin is a carcinogenic mycotoxin produced by Aspergillus flavus. Non-aflatoxigenic (Non-tox) A. flavus isolates are deployed in corn fields as biocontrol because they substantially reduce aflatoxin contamination via direct replacement and additionally via direct contact or touch with toxigenic (Tox) isolates and secretion of inhibitory/degradative chemicals. To understand touch inhibition, HPLC analysis and RNA sequencing examined aflatoxin production and gene expression of Non-tox isolate 17 and Tox isolate 53 mono-cultures and during their interaction in co-culture. Aflatoxin production was reduced by 99.7% in 72 h co-cultures. Fewer than expected unique reads were assigned to Tox 53 during co-culture, indicating its growth and/or gene expression was inhibited in response to Non-tox 17. Predicted secreted proteins and genes involved in oxidation/reduction were enriched in Non-tox 17 and co-cultures compared to Tox 53. Five secondary metabolite (SM) gene clusters and kojic acid synthesis genes were upregulated in Non-tox 17 compared to Tox 53 and a few were further upregulated in co-cultures in response to touch. These results suggest Non-tox strains can inhibit growth and aflatoxin gene cluster expression in Tox strains through touch. Additionally, upregulation of other SM genes and redox genes during the biocontrol interaction demonstrates a potential role of inhibitory SMs and antioxidants as additional biocontrol mechanisms and deserves further exploration to improve biocontrol formulations.

Aspergillus flavus originated pure compound as a potential antibacterial.

PROBLEM BACKGROUND: Penicillin was the first and most famous fungal secondary metabolite used as broad spectrum antibiotic that revolutionarised pharmaceutical research and also saved millions of lives. The over optimistic belief in 1967 that sufficient antibiotics had been discovered to defeat infectious diseases was quickly crashed with the appearance of multidrug resistant (MDR) bacteria in 1990s. This has posed a serious threat to mankind. Although scientists are making efforts to synthesize and discover new antibiotics there are not enough new drugs in pharmaceutical pipeline to beat the pace at which MDR bacteria are emerging. In view of this there is an urgent and serious medical need for new bioactive compounds to be discovered to treat infections caused by MDR pathogens. The present study is aimed to investigate the antibacterial potential of Aspergillus flavus originated compounds that may act as drug leads to treat future infections. METHODOLOGY: Among the 6 isolated fungal strains from the rhizosphere of Mentha piperetta, one was processed for isolation of secondary metabolites on the basis of preliminary antibacterial testing. Observation of morphological and microscopic features helped in identification of the fungal strain as Aspergillus flavus. Potato Dextrose Agar (PDA) medium was used for fungal growth while Czapec Yeast Broth (CYB) medium was used for production of fungal metabolites. Column chromatography technique was utilized for purification of compound from crude fungal extract and the mass of the compound was determined using Liquid Chromatography Mass Spectrometry (LCMS) method. Structure elucidation of the pure compound was performed using 500 Varian Nuclear Magnetic Resonance (NMR) machine. Docking was performed using Glide SP algorithm. Agar well diffusion method was used to determine the invitro antibacterial potential of the compound against two MDR bacterial strains i.e. Staphylococcus aureus and Proteus vulgaris. For this a total of 4 dose concentrations i.e. (100, 250, 500, 1000 µg mL- 1) of the compound were prepared and applied to bacterial strains on Mueller Hinton agar using tetracycline as control. RESULTS: The chemical name of the purified compound from A. flavus was determined as (2E)-3-[(3S, 4R)-8-hydroxy-3, 4-dimethyl-1-oxo-3, 4-dihydro-1H-2- benzopyran-7-yl] prop-2-enoic acid with the formula C14H14O5 and exact mass of 262.08. The in-Silico analysis showed that this compound has the potential to inhibit the binding pocket of S. aureus TyrRS (1JII) with docking score of - 8.67 Kcal mole- 1. The results obtained from invitro experiments were encouraging as at 1000 µg mL- 1 the compound showed 58.8% inhibition against S. aureus and 28% inhibition against P. vulgaris. CONCLUSIONS: The pure compound with formula C14H14O5 and exact mass of 262 exhibited antibacterial potential both insilico and invitro against both Gram negative and Gram positive bacteria. The compound was more active against S. aureus in comparison to P. vulgaris. From the obtained results it is concluded that this compound can be used as potent antibacterial candidate but further studies will be needed prior to its use as antibiotic.

Volatile Organic Compounds Emitted by Aspergillus flavus Strains Producing or Not Aflatoxin B1.

Aspergillus flavus is a phytopathogenic fungus able to produce aflatoxin B1 (AFB1), a carcinogenic mycotoxin that can contaminate several crops and food commodities. In A. flavus, two different kinds of strains can co-exist: toxigenic and non-toxigenic strains. Microbial-derived volatile organic compounds (mVOCs) emitted by toxigenic and non-toxigenic strains of A. flavus were analyzed by solid phase microextraction (SPME) coupled with gas chromatography-mass spectrometry (GC-MS) in a time-lapse experiment after inoculation. Among the 84 mVOCs emitted, 44 were previously listed in the scientific literature as specific to A. flavus, namely alcohols (2-methylbutan-1-ol, 3-methylbutan-1-ol, 2-methylpropan-1-ol), aldehydes (2-methylbutanal, 3-methylbutanal), hydrocarbons (toluene, styrene), furans (2,5-dimethylfuran), esters (ethyl 2-methylpropanoate, ethyl 2-methylbutyrate), and terpenes (epizonaren, trans-caryophyllene, valencene, α-copaene, ß-himachalene, γ-cadinene, γ-muurolene, δ-cadinene). For the first time, other identified volatile compounds such as α-cadinol, cis-muurola-3,5-diene, α-isocomene, and ß-selinene were identified as new mVOCs specific to the toxigenic A. flavus strain. Partial Least Square Analysis (PLSDA) showed a distinct pattern between mVOCs emitted by toxigenic and non-toxigenic A. flavus strains, mostly linked to the diversity of terpenes emitted by the toxigenic strains. In addition, the comparison between mVOCs of the toxigenic strain and its non-AFB1-producing mutant, coupled with a semi-quantification of the mVOCs, revealed a relationship between emitted terpenes (ß-chamigrene, α-corocalene) and AFB1 production. This study provides evidence for the first time of mVOCs being linked to the toxigenic character of A. flavus strains, as well as terpenes being able to be correlated to the production of AFB1 due to the study of the mutant. This study could lead to the development of new techniques for the early detection and identification of toxigenic fungi.

Neuroprotective Effect of Cyclo-(L-Pro-L-Phe) Isolated from the Jellyfish-Derived Fungus Aspergillus flavus.

Peroxisome proliferator-activated receptor (PPAR) expression has been implicated in pathological states such as cancer, inflammation, diabetes, and neurodegeneration. We isolated natural PPAR agonists-eight 2,5-diketopiperazines-from the jellyfish-derived fungus Aspergillus flavus. Cyclo-(L-Pro-L-Phe) was the most potent PPAR-γ activator among the eight 2,5-DKPs identified. Cyclo-(L-Pro-L-Phe) activated PPAR-γ in Ac2F rat liver cells and SH-SY5Y human neuroblastoma cells. The neuroprotective effect of this partial PPAR-γ agonist was examined using the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, lactate dehydrogenase release, and the Hoechst 33342 staining assay in SH-SY5Y cells. Our findings revealed that cyclo-(L-Pro-L-Phe) reduced hydrogen peroxide-induced apoptosis as well as the generation of reactive oxygen species. Rhodamine 123 staining and western blotting revealed that cyclo-(L-Pro-L-Phe) prevented the loss of mitochondrial membrane potential and inhibited the activation of mitochondria-related apoptotic proteins, such as caspase 3 and poly (ADP-ribose) polymerase. Moreover, cyclo-(L-Pro-L-Phe) inhibited the activation and translocation of nuclear factor-kappa B. Thus, the partial PPAR-γ agonist cyclo-(L-Pro-L-Phe) demonstrated potential neuroprotective activity against oxidative stress-induced neurodegeneration in SH-SY5Y cells.

Phomaligols F-I, polyoxygenated cyclohexenone derivatives from marine-derived fungus Aspergillus flavus BB1.

By tracing the 13C NMR resonances for carbonyls and enols, four new oxidized phomaligol derivatives, phomaligols F-I (1-4), along with seven known compounds (5-11) were isolated from the culture of the fungus Aspergillus flavus BB1 isolated from the marine shellfish Meretrix meretrix collected on Hailing Island, Yangjiang, China. The chemical structures and the absolute configurations of the new compounds were elucidated by MS, NMR, ECD, optical rotation, and 13C NMR calculations. Compounds 1 and 2 represent the first examples of phomaligol derivatives that contain an unusual bicyclic skeleton. All isolated compounds were tested for their cytotoxic activity. Among them, sporogen-AO 1 (8) showed potent inhibitory activity against the cancer cell lines A549, H1299, SK-BR-3, and HCT116 with IC50 values of 0.13, 0.78, 1.19, and 1.32 µM, respectively. Phomaligol G (2) displayed cytotoxic activity against the A549 and H1299 cell lines with IC50 values of 46.86 and 51.87 µM respectively. Additionally, phomaligol H (3) demonstrated cytotoxic activity against the A549 cell line with an IC50 value of 65.53 µM. Mechanistic studies of compound 8 showed that it induced apoptosis of HCT116 cells in a dose-dependent manner.

Selection and characterization of two monoclonal antibodies specific for the Aspergillus flavus major antigenic cell wall protein Aflmp1.

Aspergillus flavus is a major fungal pathogen of plants and an opportunistic pathogen of humans. In addition to the direct impact of infection, it produces immunosuppressive and carcinogenic aflatoxins. The early detection of A. flavus is therefore necessary to diagnose and monitor fungal infection, to prevent aflatoxin contamination of food and feed, and for effective antifungal therapy. Aspergillus-specific monoclonal antibodies (mAbs) are promising as diagnostic and therapeutic reagents for the tracking and treatment of Aspergillus infections, respectively. However, A. flavus has a complex cell wall composition and dynamic morphology, hindering the discovery of mAbs with well-characterized targets. Here we describe the generation and detailed characterization of mAb5.52 (IgG2aκ) and mAb17.15 (IgG1κ), which bind specifically to the highly immunogenic cell wall antigen A. flavus mannoprotein 1 (Aflmp1). Both mAbs were generated using hybridoma technology following the immunization of mice with a recombinant truncated version of Aflmp1 (ExD, including the homologous CR4 domain) produced in bacteria. We show that mAb5.52 and mAb17.15 bind specifically to A. flavus and A. parasiticus cell wall fragments (CWFs), with no cross-reaction to CWFs from other fungal pathogens. Immunofluorescence microscopy revealed that both mAbs bind to the surface of Aspergillus hyphae and that mAb17.15 also binds to spores. The epitope for both mAbs is localized within the CR4 region of the Aflmp1 protein. These Aspergillus-specific mAbs may be useful for the early detection of fungal infection in food/feed crops, for serodiagnosis in patients with invasive aspergillosis caused by A. flavus infection and for the development of antibody-expressing disease-resistant crops.

Orientated Immobilization of FAD-Dependent Glucose Dehydrogenase on Electrode by Carbohydrate-Binding Module Fusion for Efficient Glucose Assay.

The discovery or engineering of fungus-derived FAD-dependent glucose 1-dehydrogenase (FAD-GDH) is especially important in the fabrication and performance of glucose biosensors. In this study, a novel FAD-GDH gene, phylogenetically distantly with other FAD-GDHs from Aspergillus species, was identified. Additionally, the wild-type GDH enzyme, and its fusion enzyme (GDH-NL-CBM2) with a carbohydrate binding module family 2 (CBM2) tag attached by a natural linker (NL), were successfully heterogeneously expressed. In addition, while the GDH was randomly immobilized on the electrode by conventional methods, the GDH-NL-CBM2 was orientationally immobilized on the nanocellulose-modified electrode by the CBM2 affinity adsorption tag through a simple one-step approach. A comparison of the performance of the two electrodes demonstrated that both electrodes responded linearly to glucose in the range of 0.12 to 40.7 mM with a coefficient of determination R2 > 0.999, but the sensitivity of immobilized GDH-NL-CBM2 (2.1362 × 10-2 A/(M*cm2)) was about 1-fold higher than that of GDH (1.2067 × 10-2 A/(M*cm2)). Moreover, a lower detection limit (51 µM), better reproducibility (<5%) and stability, and shorter response time (≈18 s) and activation time were observed for the GDH-NL-CBM2-modified electrode. This facile and easy immobilization approach used in the preparation of a GDH biosensor may open up new avenues in the development of high-performance amperometric biosensors.

[Studies on Elastase and Elastase Inhibitor from Aspergillus flavus].

The biological properties of elastase and Aspergillus flavus elastase inhibitor (AFLEI) from A. flavus were examined. Pathogenicity of elastase was investigated in mice immunocompromised with cyclophosphamide, cyclosporine, prednisolone and carrageenan. Compared to cyclophosphamide immunocompromised mice treated with the spores of elastase nonproducing strain, cyclophosphamide immunocompromised mice treated with the spores of elastase producing strain had a significantly shorter survival rate. Molecular mass of AFLEI was determined to be 7525.8 Da. The elastolytic activity of elastases from A. flavus, and human leukocytes were inhibited by AFLEI. The primary structure of AFLEI was determined by the Edman sequencing procedure. The search for amino acid homology with other proteins demonstrated that amino acid residues 1 to 68 of AFLEI are 100% identical to residues 20 to 87 of the hypothetical protein AFUA_3G14940 of A. fumigatus. When immunocompromised mice administered of cyclophosphamide were infected by inhalation of A. flavus then administered amphotericin B (AMPH) alone or in combination with AFLEI, survival rate tended to be higher with combination treatment than with AMPH alone. Moreover, although extensive bleeding was seen in pathology sections taken from rat lung resected 24 h after elastase was administered to the lung via the bronchus, this bleeding was inhibited by AFLEI. The X-ray analysis has revealed that the structure of this inhibitor was wedge shaped and composed of a binding loop and a scaffold protein core. As synthetic-inhibitor strongly inhibited cytotoxicity induced by elastase in human-derived cells, it could prove beneficial for the treatment of pulmonary aspergillosis.

The AFLATOX® Project: Approaching the Development of New Generation, Natural-Based Compounds for the Containment of the Mycotoxigenic Phytopathogen Aspergillus flavus and Aflatoxin Contamination.

The control of the fungal contamination on crops is considered a priority by the sanitary authorities of an increasing number of countries, and this is also due to the fact that the geographic areas interested in mycotoxin outbreaks are widening. Among the different pre- and post-harvest strategies that may be applied to prevent fungal and/or aflatoxin contamination, fungicides still play a prominent role; however, despite of countless efforts, to date the problem of food and feed contamination remains unsolved, since the essential factors that affect aflatoxins production are various and hardly to handle as a whole. In this scenario, the exploitation of bioactive natural sources to obtain new agents presenting novel mechanisms of action may represent a successful strategy to minimize, at the same time, aflatoxin contamination and the use of toxic pesticides. The Aflatox® Project was aimed at the development of new-generation inhibitors of aflatoxigenic Aspergillus spp. proliferation and toxin production, through the modification of naturally occurring molecules: a panel of 177 compounds, belonging to the thiosemicarbazones class, have been synthesized and screened for their antifungal and anti-aflatoxigenic potential. The most effective compounds, selected as the best candidates as aflatoxin containment agents, were also evaluated in terms of cytotoxicity, genotoxicity and epi-genotoxicity to exclude potential harmful effect on the human health, the plants on which fungi grow and the whole ecosystem.

Chromolaena laevigata (Asteraceae) as a source of endophytic non-aflatoxigenic Aspergillus flavus: chemical profile in different culture conditions and biological applications.

Endophytes are microorganisms that form symbiotic relationships with their host. These microorganisms can produce a variety of secondary metabolites, some of which have inhibitory effects on pests and pathogens or even act to promote plant growth. Due to these characteristics, these microorganisms are used as sources of biologically active substances for a wide range of biotechnological applications. Based on that, the aim of this study was to evaluate the production of metabolites of the endophytic Aspergillus flavus CL7 isolated from Chromolaena laevigata, in four different cultivation conditions, and to determine the antimicrobial, cytotoxic, antiviral, and antioxidant potential of these extracts. The multiphasic approach used to identify this strain was based on morphology and ITS gene sequence analysis. The chemical investigation of A. flavus using potato dextrose and minimal medium, using both stationary and agitated methods, resulted in the isolation of kojic acid, α-cyclopiazonic acid, and 20,25-dihydroxyaflavinine. Another 18 compounds in these extracts were identified by UHPLC-HRMS/MS, of which dideacetyl parasiticolide A has been described for the first time from A. flavus. Aflatoxins, important chemomarkers of A. flavus, were not detected in any of the extracts, thus indicating that the CL7 strain is non-aflatoxigenic. The biological potential of all extracts was evaluated, and the best results were observed for the extract obtained using minimal medium against Trichophyton rubrum and Mycobacterium tuberculosis.

Asperorydines N-P, three new cyclopiazonic acid alkaloids from the marine-derived fungus Aspergillus flavus SCSIO F025.

Three new tricyclic cyclopiazonic acid (CPA) related alkaloids asperorydines N-P (1-3), together with six known compounds (4-9) were isolated and characterized from the fungus Aspergillus flavus SCSIO F025 derived from the deep-sea sediments of South China Sea. The structures including absolute configurations of 1-3 were deduced from spectroscopic data, X-ray diffraction analysis, and electronic circular dichroism (ECD). All compounds were evaluated for the antioxidative activities against DPPH, cytotoxic activities against four tumor cell lines (SF-268, HepG-2, MCF-7, and A549), and antimicrobial activities. Compound 9 showed significant radical scavenging activities against DPPH with an IC50 value of 62.23 µM and broad-spectrum cytotoxicities against four tumor cell lines with IC50 values ranging from 24.38 to 48.28 µM. Furthermore, compounds 4-9 exhibited weak antimicrobial activities against E scherichia coli, and compound 9 also showed antibacterial activity against Bacillus thuringiensis, Micrococcus lutea, Staphylococcus aureus, Bacillus subtilis, Methicillin resistant Staphylococcus aureus.

Computational modelling of survival of Aspergillus flavus in peanut kernels during hot air-assisted radio frequency pasteurization.

In recent years, radio frequency (RF) heating is getting popular as an alternative pasteurization method for agricultural commodities and low moisture foods. Computer simulation is an effective way to help understand RF interactions with food components and predict temperature distributions among food samples after RF treatments. In this study, a computer model based on Joule heating and thermal inactivation kinetic of A. flavus was established to predict both temperature distribution and microbial reduction among peanut kernels after RF processing. For the process validation, three 2-g peanut samples inoculated with 40 µL A. flavus were placed at three representative locations among 2.17 kg peanut kernels and subjected to various processing conditions in a 27.12 MHz, 6 kW RF heating unit together with hot air system. Results showed that the average difference of the sample temperature and microbial reduction between simulation and experiment was small with RMSE values of 0.009 °C and 0.012 °C, and 0.31 log CFU/g and 0.42 log CFU/g for peanut moisture contents of 7.56% and 12.02% w. b., respectively. Nonuniform RF heating resulted in the least lethality of A. flavus at the cold spot. The validated computer model was further used to estimate microbial reduction distributions at other target temperatures based on predicted temperature profiles. This computer model may help design the RF pasteurization protocols for peanut kernels without extensive experiments in food industry.

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.

Distribution of active ingredients of a commercial aflatoxin biocontrol product in naturally occurring fungal communities across Kenya.

Human populations in Kenya are repeatedly exposed to dangerous aflatoxin levels through consumption of contaminated crops. Biocontrol with atoxigenic Aspergillus flavus is an effective method for preventing aflatoxin in crops. Although four atoxigenic A. flavus isolates (C6E, E63I, R7H and R7K) recovered from maize produced in Kenya are registered as active ingredients for a biocontrol product (Aflasafe KE01) directed at preventing contamination, natural distributions of these four genotypes prior to initiation of commercial use have not been reported. Distributions of the active ingredients of KE01 based on haplotypes at 17 SSR loci are reported. Incidences of the active ingredients and closely related haplotypes were determined in soil collected from 629 maize fields in consecutive long and short rains seasons of 2012. The four KE01 haplotypes were among the top ten most frequent. Haplotype H-1467 of active ingredient R7K was the most frequent and widespread haplotype in both seasons and was detected in the most soils (3.8%). The four KE01 haplotypes each belonged to large clonal groups containing 27-46 unique haplotypes distributed across multiple areas and in 21% of soils. Each of the KE01 haplotypes belonged to a distinct vegetative compatibility group (VCG), and all A. flavus with haplotypes matching a KE01 active ingredient belonged to the same VCG as the matching active ingredient as did all A. flavus haplotypes differing at only one SSR locus. Persistence of the KE01 active ingredients in Kenyan agroecosystems is demonstrated by detection of identical SSR haplotypes six years after initial isolation. The data provide baselines for assessing long-term influences of biocontrol applications in highly vulnerable production areas of Kenya.

Chemical repertoire and biosynthetic machinery of the Aspergillus flavus secondary metabolome: A review.

Filamentous fungi represent a rich source of extrolites, including secondary metabolites (SMs) comprising a great variety of astonishing structures and interesting bioactivities. State-of-the-art techniques in genome mining, genetic manipulation, and secondary metabolomics have enabled the scientific community to better elucidate and more deeply appreciate the genetic and biosynthetic chemical arsenal of these microorganisms. Aspergillus flavus is best known as a contaminant of food and feed commodities and a producer of the carcinogenic family of SMs, aflatoxins. This fungus produces many SMs including polyketides, ribosomal and nonribosomal peptides, terpenoids, and other hybrid molecules. This review will discuss the chemical diversity, biosynthetic pathways, and biological/ecological role of A. flavus SMs, as well as their significance concerning food safety and security.

Development and validation of TOF/Q-TOF MS/MS, HPLC method and in vitro bio-strategy for aflatoxin mitigation.

Some secondary metabolites produced by fungi are carcinogenic, hepatotoxic, and/or cause birth defects in humans and animals. We developed and optimised bio-analytical tools for detection of metabolites, aflatoxins and evaluated the effectiveness of the methods in co-infected maize tissues. Isolate KSM012 (atoxigenic) demonstrated no peaks and no blue fluorescence on HPLC and TLC plates respectively confirming non-toxicity. AFB1 and AFB2 were produced by Isolate KSM015 in addition to AFG1 and AFG2, which is an indication of possible SBG morphotype. The limits of quantification and detection ranged from 0.02 to 35.81 µg/mL and 0.01-6.8 µg/mL, respectively. The best mass spectrum with lowest noise was obtained at 100% ACN and sterile water spiked with 0.1% formic acid at a flow rate of 0.3 mL/min. The positive ion mode with electrospray ionisation application exhibited better fragmentation for mycotoxins. In total 17 metabolites were detected by targeted and formula mass. KDVI maize line exhibited high fungal colonisation in comparison to GAF4 at equal co-infection ratio 50:50. AFB1 and AFG2 were remarkably higher in GAF4 in comparison to sensitive KDV1 (p ˂ 0.05). The detection limits, linearity and sensitivity showed the method developed was suitable for the determination of mycotoxin in comparisons to the guidelines of European Commission 657/EC 2002.

Facile dispersive solid-phase extraction based on humic acid for the determination of aflatoxins in various edible oils.

Aflatoxins (AFs), as the secondary metabolites of the toxigenic fungi Aspergillus flavus and Aspergillus parasiticus, are well known to be extremely harmful to humans and animals because of their high toxicity, mutagenicity, carcinogenicity, and teratogenicity. Recurring and increasing studies on AF ingestion incidents indicate that AF contamination is a serious food safety issue worldwide. Currently, immunoaffinity chromatography (IAC) has become the most conventional sample clean-up method for determining AFs in foodstuffs. However, the IAC method may be limited to some laboratories because it requires the use of expensive disposable cartridges and the IA procedure is time-consuming. Herein, to achieve the cost-effective determination of AFs in edible oils, we developed a dispersive solid-phase extraction (DSPE) clean-up method based on humic acids (HAs), which is followed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis. HAs could be directly used as a DSPE sorbent after simple treatment without any chemical modification. In the HA-DSPE, AFs could remain on the HA sorbent by both hydrophobic and hydrophilic interactions, whereas the oil matrix was retained on HA via only hydrophobic interactions. The oil matrix could be sufficiently washed off by n-hexane, whereas the AFs could still be retained on HA; thus, the selective extraction of AFs and clean-up of oil matrices were achieved. Under the optimal conditions of HA-DSPE, satisfactory recoveries ranging from 81.3% to 106.2% for four AFs (B1, B2, G1, and G2) were achieved in various oil matrices i.e. blended oil, mixed olive oil, tea oil, sunflower seed oil, rapeseed oil, sesame oil, soybean oil, rice oil, corn oil, and peanut oil. Minor matrix effects ranging from 89.3% to 112.9% were obtained for the four AFs, which were acceptable. Moreover, the LODs of AFs between 0.063 and 0.102 µg kg-1 completely meet the regulatory levels fixed by the Food and Drug Administration (FDA), the European Union (EU), China, or other countries. The proposed methodology was further validated using a naturally contaminated peanut oil, and the results indicated that the accuracy of the HA-DSPE could match the accuracy of the referenced IAC. In addition, HA-DSPE can be used to directly treat diluted edible oil without liquid-liquid extraction and HA is cheap and can be easily obtained from the market worldwide; these advantages make the proposed methodology simple, low-cost, and accessible for the determination of AFs in edible oils.

Asperienes A-D, Bioactive Sesquiterpenes from the Marine-Derived Fungus Aspergillus flavus.

Marine-derived fungi of the genera Aspergillus could produce novel compounds with significant bioactivities. Among these fungi, the strain Aspergillus flavus is notorious for its mutagenic mycotoxins production. However, some minor components with certain toxicities from A. flavus have not been specifically surveyed and might have potent biological activities. Our investigation of the marine-derived fungus Aspergillus flavus CF13-11 cultured in solid medium led to the isolation of four C-6'/C-7' epimeric drimane sesquiterpene esters, asperienes A-D (1-4). Their absolute configurations were assigned by electronic circular dichroism (ECD) and Snatzke's methods. This is the first time that two pairs of C-6'/C-7' epimeric drimane sesquiterpene esters have successfully been separated. Aperienes A-D (1-4) displayed potent bioactivities towards four cell lines with the IC50 values ranging from 1.4 to 8.3 µM. Interestingly, compounds 1 and 4 exhibited lower toxicities than 2 and 3 toward normal GES-1 cells, indicating more potential for development as an antitumor agent in the future.