Reduce Aflatoxin Contamination Research Articles

Biocontrol Strains Differentially Shift the Genetic Structure of Indigenous Soil Populations of Aspergillus flavus.

Biocontrol using non-aflatoxigenic strains of Aspergillus flavus has the greatest potential to mitigate aflatoxin contamination in agricultural produce. However, factors that influence the efficacy of biocontrol agents in reducing aflatoxin accumulation under field conditions are not well-understood. Shifts in the genetic structure of indigenous soil populations of A. flavus following application of biocontrol products Afla-Guard and AF36 were investigated to determine how these changes can influence the efficacy of biocontrol strains in reducing aflatoxin contamination. Soil samples were collected from maize fields in Alabama, Georgia, and North Carolina in 2012 and 2013 to determine changes in the population genetic structure of A. flavus in the soil following application of the biocontrol strains. A. flavus L was the most dominant species of Aspergillus section Flavi with a frequency ranging from 61 to 100%, followed by Aspergillus parasiticus that had a frequency of <35%. The frequency of A. flavus L increased, while that of A. parasiticus decreased after application of biocontrol strains. A total of 112 multilocus haplotypes (MLHs) were inferred from 1,282 isolates of A. flavus L using multilocus sequence typing of the trpC, mfs, and AF17 loci. A. flavus individuals belonging to the Afla-Guard MLH in the IB lineage were the most dominant before and after application of biocontrol strains, while individuals of the AF36 MLH in the IC lineage were either recovered in very low frequencies or not recovered at harvest. There were no significant (P > 0.05) differences in the frequency of individuals with MAT1-1 and MAT1-2 for clone-corrected MLH data, an indication of a recombining population resulting from sexual reproduction. Population mean mutation rates were not different across temporal and spatial scales indicating that mutation alone is not a driving force in observed multilocus sequence diversity. Clustering based on principal component analysis identified two distinct evolutionary lineages (IB and IC) across all three states. Additionally, patristic distance analysis revealed phylogenetic incongruency among single locus phylogenies which suggests ongoing genetic exchange and recombination. Levels of aflatoxin accumulation were very low except in North Carolina in 2012, where aflatoxin levels were significantly (P < 0.05) lower in grain from treated compared to untreated plots. Phylogenetic analysis showed that Afla-Guard was more effective than AF36 in shifting the indigenous soil populations of A. flavus toward the non-toxigenic or low aflatoxin producing IB lineage. These results suggest that Afla-Guard, which matches the genetic and ecological structure of indigenous soil populations of A. flavus in Alabama, Georgia, and North Carolina, is likely to be more effective in reducing aflatoxin accumulation and will also persist longer in the soil than AF36 in the southeastern United States.

Pseudomonas fluorescens Pf7: A Potential Biocontrol Agent against Aspergillus flavus Induced Aflatoxin Contamination in Groundnut

Aflatoxin contamination is a qualitative problem in groundnut (Arachis hypogaea L.) occurring at both pre-and post-harvest stages. These aflatoxins are secondary metabolites produced by Aspergillus flavus and A. parasiticus and have carcinogenic, hepatotoxic, teratogenic and immuno-suppressive effects. Use of plant growth-promoting rhizobacteria (PGPR) is a viable and sustainable option in managing aflatoxin problem in groundnut. Our present study is aimed at identifying a plant growth-promoting rhizobacteria (PGPR) strain with superior antagonistic abilities on A. flavus infection, aflatoxin contamination and to determine its mode of action. Ten native P. fluorescens isolates were isolated from groundnut rhizosphere and screened against A. flavus by dual culture and in vitro seed colonization (IVSC) assays. In dual culture and IVSC studies, Pf7 exhibited higher degree of antagonism on A. flavus (54% inhibition), inhibited its colonization and reduced aflatoxin contamination (27.8 µg kg-1) in kernels.

Dual culture of atoxigenic and toxigenic strains of Aspergillus flavus to gain insight into repression of aflatoxin biosynthesis and fungal interaction

Application of atoxigenic strains to compete against toxigenic strains of Aspergillus flavus strains has emerged as one of the practical strategies for reducing aflatoxin contamination in corn, peanut, and tree nuts. The actual mechanism that results in aflatoxin reduction is not fully understood. Real-time RT-PCR and relative quantification of gene expression protocol were applied to elucidate the molecular mechanism. Transcriptional analyses of aflatoxin biosynthetic gene cluster in dual culture of toxigenic and atoxigenic A. flavus strains were carried out. Six targeted genes, aflR, aflJ, omtA, ordA, pksA, and vbs, were downregulated to variable levels depending on paired strains of toxigenic and atoxigenic A. flavus. Consistent with the decreased gene expression levels, the aflatoxin concentrations in dual cultures were reduced significantly in comparison with toxigenic cultures. Fluorescent images showed fungal hyphae in dual culture displayed green fluorescent, and contacts of live hyphae were seen. A coconut agar plate assay was used to show that toxigenic A. flavus colony produced blue fluorescence under long UV exposure, suggesting that aflatoxin is exported outside fungal hyphae. Furthermore, the assay was applied to demonstrate the potential role of thigmo-regulation in fungal interaction.

Effects of postharvest handling practices on quality of groundnuts and aflatoxin contamination

The increased cases of aflatoxin contamination are exacerbated by poor post-harvest management practices, coupled with adverse climatic conditions at harvest and post-harvest stages. This study therefore was carried out to improve safety and quality of groundnuts from aflatoxin contamination, through use of proper postharvest handling practices. Specifically the study determined the effects of harvesting dates and drying methods on aflatoxin contamination. Field experiments were carried out both at Chitedze and Chitala Agricultural Research Stations in Malawi during 2017/2018 growing season. A randomized complete block design in a split plot arrangement with three harvesting dates as the main plot and four drying methods as the sub-plots replicated three times was used. Groundnut was assessed for kernel infection by Aspergillus flavus, and level of aflatoxin contamination. Significantly low levels of about 0.5μg/ Kg of A. flavus infection and aflatoxin contamination were observed at 90 days after sowing (DAS). Higher aflatoxin contamination of up to 5μg/ Kg was observed at 80 DAS, and 10 days late after physiological maturity (100 DAS). This study also identified Mandela cock, aframe drying rack as effective drying method that can reduce aflatoxin contamination in groundnuts by 75 %. Moreover, Mandela cock drying method was shown as the most effective compared to A-frame and drying rack drying method. Current study therefore recommends for adoption of timely harvesting at physiological maturity, and drying using either Mandela cock or A-frame and drying rack. Further studies need to be carried on biological control of aflatoxin contamination.

Post-harvest practices for aflatoxin control: Evidence from Kenya

We assess the impact of a package of post-harvest technologies on aflatoxin contamination of maize through a randomized trial in rural Kenya. Some elements of this package (training and provision of plastic sheets for sun-drying) were provided free of charge to all participants in treatment villages and were widely adopted. Others (a mobile drying service and hermetic storage bags) were provided free to a subset of randomly selected farmers in treatment villages while others had to pay. Overall, the intervention reduced aflatoxin contamination by over 50%. Most of this reduction appears to be due training and the use of drying sheets, the lowest-cost of all the technologies offered.

Set3 Is Required for Asexual Development, Aflatoxin Biosynthesis, and Fungal Virulence in Aspergillus flavus.

Aspergillus flavus is an opportunistic pathogenic fungus for both plant and animal that produces carcinogenic toxins termed aflatoxins (AFs). To identify possible genetic targets to reduce AF contamination, in this study, we have characterized a novel A. flavus Set3, and it shares sequence homology with the yeast protein Set3. The set3 deletion mutants present no difference in growth rate but alterations in asexual development and secondary metabolite production when compared to the A. flavus wild type. Specifically, deletion of set3 gene decreases conidiophore formation and conidial production through downregulating expression of brlA and abaA genes. In addition, normal levels of set3 are required for sclerotial development and expression of sclerotia-related genes nsdC and sclR. Further analyses demonstrated that Set3 negatively regulates AF production as well as the concomitant expression of genes in the AF gene cluster. Importantly, our results also display that A. flavus Set3 is involved in crop kernel colonization. Taking together, these results reveal that a novel Set3 plays crucial roles in morphological development, secondary metabolism, and fungal virulence in A. flavus.

Potential application of lactic acid starters in the reduction of aflatoxin contamination in fermented sorghum-millet beverages

BackgroundAflatoxin contamination in traditionally fermented cereal-based beverages is a serious food safety challenge considering that commercialization of these products is rising. The challenge is aggravated by the fact that aflatoxin elimination from the food chain is almost impossible. This paper focuses on Obushera, a popular traditional spontaneously fermented sorghum – millet beverage from Uganda.MethodMold and total aflatoxin levels in cereal flours and Obushera from different markets in Kampala were determined. Ability of lactic acid bacteria (LAB) starters from Obushera; Lb. plantarum MNC 21, W. confusa MNC 20 and L. lactis MNC 24 to bind aflatoxin B1 (AFB1) was evaluated against Lb. rhamnosus yoba 2012 as the reference strain.ResultsMold counts in sorghum, millet and Obushera ranged between 0.0–2.4 log cfu/g, 2.0–6.5 log cfu/g and 2.0–5.5 log cfu/g, respectively. None of the flours complied with food safety standards for molds (maximum = 4 log cfu/g) while 88.0% of Obushera did (standard = maximum 1.3 log cfu/g). Total aflatoxin levels in sorghum, millet and Obushera were 22.3 ± 21.2 ppb, 9.9 ± 10.0 ppb and 10.4 ± 6.1 ppb, respectively. LAB bound 19.3–69.4% of AFB1 in a 1000 ppb solution with binding efficiency in the order of Lb. rhamnosus yoba 2012 = Lb. plantarum MNC 21 > W. confusa MNC 20 = L. lactis MNC 24. The LAB-AFB1complex remained stable under a series of washes with physiological saline.ConclusionThis study indicates that LAB with aflatoxin binding properties can be exploited for controlled cereal fermentations to improve food safety.

Atoxigenic Aspergillus flavus Isolates Endemic to Almond, Fig, and Pistachio Orchards in California with Potential to Reduce Aflatoxin Contamination in these Crops.

In California, aflatoxin contamination of almond, fig, and pistachio has become a serious problem in recent years due to long periods of drought and probably other climatic changes. The atoxigenic biocontrol product Aspergillus flavus AF36 has been registered for use to limit aflatoxin contamination of pistachio since 2012 and for use in almond and fig since 2017. New biocontrol technologies employ multiple atoxigenic genotypes because those provide greater benefits than using a single genotype. Almond, fig, and pistachio industries would benefit from a multi-strain biocontrol technology for use in these three crops. Several A. flavus vegetative compatibility groups (VCGs) associated with almond, fig, and pistachio composed exclusively of atoxigenic isolates, including the VCG to which AF36 belongs to, YV36, were previously characterized in California. Here, we report additional VCGs associated with either two or all three crops. Representative isolates of 12 atoxigenic VCGs significantly (P < 0.001) reduced (>80%) aflatoxin accumulation in almond and pistachio when challenged with highly toxigenic isolates of A. flavus and A. parasiticus under laboratory conditions. Isolates of the evaluated VCGs, including AF36, constitute valuable endemic, well-adapted, and efficient germplasm to design a multi-crop, multi-strain biocontrol strategy for use in tree crops in California. Availability of such a strategy would favor long-term atoxigenic A. flavus communities across the affected areas of California, and this would result in securing domestic and export markets for the nut crop and fig farmer industries and, most importantly, health benefits to consumers.

Aflatoxin B1 (AFB1) production by Aspergillus flavus and Aspergillus parasiticus on ground Nyjer seeds: The effect of water activity and temperature

Nyjer oil seed cake supports high levels of aflatoxin B1 (AFB1) production. AFB1 is a secondary metabolite of Aspergillus flavus and A. parasiticus, classified as a Class 1A carcinogen. The aim of this study was to determine the effects of temperature (20, 27, and 35 °C) and water activity (0.82, 0.86, 0.90, 0.94, and 0.98 aw) on fungal growth and AFB1 production of A. flavus and A. parasiticus on ground Nyjer seeds over a 30-day incubation period.Linear regression models indicated that both fungal growth and AFB1 production were significantly influenced by water activity of Nyjer seeds and incubation temperature. The two fungi did not grow on Nyjer seeds at 0.82 aw at the three incubation temperatures. The most favorable growth conditions for both fungi were 0.90–0.98 aw at 27 °C or 0.90–0.94 aw at 35 °C. The optimum temperature for AFB1 production was 27 °C for both A. flavus and A. parasiticus (with regression coefficients of 6.01 and 9.11, respectively). Both fungi were likely to produce high levels of AFB1 at 0.90 aw (with regression coefficients of 3.56 for A. flavus and 7.17 for A. parasiticus). Aspergillus flavus only produced AFB1 on seeds with 0.90–0.98 aw at 27 °C (in the range of 203–282 μg/kg) and on seeds with 0.90 aw at 35 °C (212 μg/kg). No detectable AFB1 was produced by this fungus in any other culture conditions that were studied.Aspergillus parasiticus, in contrast, was able to produce AFB1 under all of the growth conditions. At 20 °C, this fungus produced the highest level of AFB1 (212 μg/kg) at high water activity (0.98 aw). At 27 °C, A. parasiticus produced high levels of AFB1 (in the range of 209–265 μg/kg) at a wide range of water activities (0.86–0.98 aw). In the entire study, the highest AFB1 concertation for A. parasiticus was detected on seeds incubated at high temperature (35 °C) and low water activity (0.86 aw). The findings of this study could help optimize the storage conditions of Nyjer oil seeds to reduce aflatoxin contamination.

Aflatoxin in maize: a review of the early literature from "moldy-corn toxicosis" to the genetics of aflatoxin accumulation resistance.

Aflatoxin is a potent toxin produced by Aspergillus flavus Link:Fr, an opportunistic ear-rot pathogen of maize (Zea mays L. subsp. Mays). Prior to the discovery of aflatoxin, A. flavus was considered a minor pathogen and was not a priority for maize breeders or pathologists. Aflatoxin was discovered in England in 1961 following an epidemic in poultry. By the early 1970s, surveys of agricultural commodities in the USA found that maize produced in the Southeast was especially vulnerable to aflatoxin contamination. Aflatoxin contamination was initially treated as a post-harvest issue, but pre-harvest contamination was proven by 1975. Pre-harvest contamination meant that genetically based host-plant resistance was a possible solution. The potential magnitude of the problem became apparent in 1977 when the southeastern US maize crop suffered epidemic aflatoxin contamination. The first experiment demonstrating the heritability of host-plant resistance to aflatoxin accumulation was published in 1978. These events combined to make breeding for reduced aflatoxin contamination both a high priority and a rational breeding objective. This review surveys the early scientific literature in order to place research on the genetics of aflatoxin accumulation in maize into historical context. It tells the story of how multi-disciplinary research began with veterinary diseases of unknown etiology and resulted in host-plant resistance to a previously minor plant pathogen becoming a central public sector breeding objective.

Provitamin A Carotenoids in Grain Reduce Aflatoxin Contamination of Maize While Combating Vitamin A Deficiency.

Aflatoxin contamination of maize grain and products causes serious health problems for consumers worldwide, and especially in low- and middle-income countries where monitoring and safety standards are inconsistently implemented. Vitamin A deficiency (VAD) also compromises the health of millions of maize consumers in several regions of the world including large parts of sub-Saharan Africa. We investigated whether provitamin A (proVA) enriched maize can simultaneously contribute to alleviate both of these health concerns. We studied aflatoxin accumulation in grain of 120 maize hybrids formed by crossing 3 Aspergillus flavus resistant and three susceptible lines with 20 orange maize lines with low to high carotenoids concentrations. The hybrids were grown in replicated, artificially-inoculated field trials at five environments. Grain of hybrids with larger concentrations of beta-carotene (BC), beta-cryptoxanthin (BCX) and total proVA had significantly less aflatoxin contamination than hybrids with lower carotenoids concentrations. Aflatoxin contamination had negative genetic correlation with BCX (-0.28, p < 0.01), BC (-0.18, p < 0.05), and proVA (-0.23, p < 0.05). The relative ease of breeding for increased proVA carotenoid concentrations as compared to breeding for aflatoxin resistance in maize suggests using the former as a component of strategies to combat aflatoxin contamination problems for maize. Our findings indicate that proVA enriched maize can be particularly beneficial where the health burdens of exposure to aflatoxin and prevalence of VAD converge with high rates of maize consumption.

Antifungal activities of coating incorporated with Saccharomyces cerevisiae cell wall mannoprotein on Aspergillus flavus growth and aflatoxin production in pistachio (Pistacia vera L.)

AbstractIn this study, the effects of the mannoprotein isolated from Saccharomyces cerevisiae (ScMnn) were evaluated against Aspergillus flavus growth and aflatoxin (AF) production in pistachios. The mannoprotein extracted from the cell wall of S. cerevisiae was incorporated in gelatin‐based solution at different concentration (0, 0.5, 1 and 1.5% w/w) then used as coating of kernels. A. flavus spore suspension (104 spores/mL) was inoculated on both coated (cp) and non‐coated (ncp) pistachios. In cp samples, the mycelium development was delayed and AF content decreased, compared with the ncp ones. Noteworthy, the coating containing 1.5% ScMnn decreased AFB1 up to 42.81%. The ScMnn could be considered as an operative agent to reduce A. flavus growth and AF contamination in pistachios. The edible and food grade properties are important in its application as novel additive and as bioactive agent in coatings for the control of AF in pistachios.Practical applicationsActive edible coating containing Saccharomyces cerevisiae mannoprotein could be applied in pistachio industry to control Aspergillus flavus growth and reduce aflatoxin contamination. This antifungal coating could improve the safety of pistachio nuts.

Evaluation of maize inbred lines and topcross progeny for resistance to pre-harvest aflatoxin contamination

Pre-harvest aflatoxin contamination occurs in maize following kernel colonization by Aspergillus flavus. Aflatoxin contamination resistance is a highly desired trait in maize breeding programs. The identification of novel sources of resistance to pre-harvest aflatoxin contamination is a major focus in germplasm screening efforts. Here, we performed a field evaluation of 64 inbred lines over two years for pre-harvest aflatoxin contamination. Topcrosses were also performed with two testers, B73 and Mo17, to generate 128 F1 hybrids which were also evaluated over two years. Hybrid performance was used to calculate both general combining ability (GCA) of the inbreds, and observed heterosis for aflatoxin resistance. Over both years of the study, aflatoxin concentrations ranged from 80 ± 47 to 17,617 ± 8816 μg kg−1 for inbreds, and from 58 ± 39 to 2771 ± 780 μg kg−1 for hybrids with significant variation between years and lines. The inbred lines CML52, CML69, CML247, GT-603, GEMS-0005, Hi63, Hp301, and M37 W showed <1000 μg kg−1 of aflatoxin accumulation in both years of the study and less than the resistant check, Mp313E, in at least one season. Among these, CML52, GT-603, and Hi63 also showed significant GCA with the testers in hybrid progeny. CML52, GT-603, and M37 W also showed heterotic effects of −13.64%, −12.47%, and − 24.50%, respectively, with B73 resulting in reduced aflatoxin contamination. GT-603 also showed a similar heterotic effect for aflatoxin contamination, −13.11%, with Mo17 indicating that this line may serve as a versatile source of aflatoxin contamination resistance in breeding programs.

Occurrence of aflatoxin in agricultural produce from localmarkets in Burundi and Eastern Democratic Republic ofCongo.

Aflatoxins are noxious secondary metabolites, of certain fungal species, found in food and feed. Contamination of a commodity with aflatoxins is associated with production and storage losses, and subsequently less food availability. Aflatoxins can also pose human health risks and represent a barrier to the development of trade, in both domestic and international markets. In this study, samples of cassava, maize, groundnut, beans, soybean, sorghum and milk, and their processed products were collected from local markets in Burundi and Eastern DRC. In order to investigate the levels of aflatoxin, crop samples were analyzed using a single step lateral flow immunochromatographic assay (Reveal Q+), while enzyme‐linked immune‐sorbent assay (ELISA) was used to analyze aflatoxin‐M1 in milk, yogurt, and cheese samples. The results revealed the presence of aflatoxins in all samples from both countries, with levels ranging from 1.3 to 2,410 μg/kg. Samples collected from Burundi contained relatively higher (p > 0.0.5) levels of aflatoxins. In 51% of all the crops samples, total aflatoxin contamination was above the EU maximum tolerable level of 4 μg/kg. Processed products, particularly from groundnut, maize, and sorghum, had the highest levels of aflatoxin contamination when compared to unprocessed grain. With regard to milk and dairy products, the level of aflatoxin‐M1 ranged from 4.8 to 261.1 ng/kg. Approximately 29% of milk and yogurt samples had aflatoxin‐M1 higher than the EU regulatory limit of 50 ng/kg, whereas 20% of cheese samples were found to be contaminated at levels higher than the maximum limit of 250 ng/kg. These results can serve as the basis for pre‐ and postharvest approaches to reduce aflatoxin contamination in agricultural commodities in Burundi and Eastern DRC in order to reduce health risk, avoid reduced production in livestock, and open up export markets.

Aflatoxin awareness and Aflasafe adoption potential of Nigerian smallholder maize farmers: OPEN ACCESS.

Aflatoxin is a potent mycotoxin that can cause cancer and death and is associated with stunted growth. Prevalence of aflatoxin is widespread in Africa negatively impacting health and trade. Aflasafe is a biological control product that can be applied to maize or groundnut fields to reduce aflatoxin contamination. This study examines the levels of aflatoxin and Aflasafe awareness and understanding among smallholder maize farmers in Nigeria. In addition, the factors affecting Aflasafe purchase patterns and sustained usage over multiple growing seasons by farmers were evaluated. In-person surveys of 902 Nigerian smallholder farmers were conducted during October and November of 2016. This work contributes to the existing literature by documenting awareness levels of aflatoxin and use of Aflasafe as a control in Nigeria. Results suggest that the level of awareness of aflatoxin was very high in states where Aflasafe was promoted as an intervention for aflatoxin management. In Kaduna state, the region with the longest intervention, there was a consistent increase in the usage of Aflasafe since its introduction in 2010. Furthermore, farmers who purchase Aflasafe bundled (combined) with other inputs were more likely to persist in using the product. Education was found to significantly and positively impact continued usage of Aflasafe. Continued interventions, promotion and general education of the public are recommended for increased awareness, trial, and adoption of Aflasafe in Nigeria.

Incidence and farmers' knowledge of aflatoxin contamination and control in Eastern Democratic Republic of Congo.

Despite efforts to reduce aflatoxin contamination and associated mycotoxin poisoning, the phenomenon continues to pose a public health threat in food and feed commodity chains. In this study, 300 samples of cassava, maize, and groundnut were collected from farmers’ households in Eastern DRC and analyzed for incidence of aflatoxins. In addition, the farmers’ level of knowledge of the causes and consequences of contamination and the measures for prevention were also examined by administering questionnaires to a cross section of 150 farmers. The results showed the presence of aflatoxins in all samples, with levels ranging from 1.6 to 2,270 μg/kg. In 68% of all samples, total aflatoxin contamination was above 4 μg/kg, the maximum tolerable level set by the European Union. Farmers ranked high humidity, improper storage practices, and poor soils as potential causes of aflatoxin contamination and changes in color, smell, and taste, and difficulty in selling crops as consequences. They identified crop management practices as the most effective way to control contamination. The results also revealed that most farmers apply preharvest crop management practices as a means of controlling contamination. More educated households were more knowledgeable about aflatoxins. Female‐headed and married households were less likely to be willing to pay for aflatoxin control. About 28% of farmers claimed to be willing to allocate resources to seed intervention while a smaller proportion agreed to pay for training and information services. The result further suggests that an adoption of pre‐ and postharvest technologies together with awareness creation is still required to reduce aflatoxin contamination in the country.

Impact of Insect Densities Tribolium Castaneum on the Benzoquinone Secretions and Aflatoxins Levels in Wheat Flour During Storage Periods

Objectives: The present study was prepared to investigate the impact of insect density, adult emergence of Tribolium castaneum on the secretion of Benzoquinones (BQs) consist of methyl-1,4-benzoquinone(MBQ) and Ethyl-1,4-Benzoquinone (EBQ), and accumulation of Aflatoxins (AFs) in wheat flour stored at different periods. Methods: Forty grams of wheat flour were put into small glass jars (8 cm diameter and 12 cm length). Then T. castaneum was put in each jars at rates of 10, 20 and 30 unsexed pairs of insect adult. The jars were covered with muslin cloth and the rubber band was fixed to prevent insects to escape. A glass jar without any insects served as the control. The jars lifted on bench in the laboratory for two, three and four months of storage under laboratory temperature conditions (with average 28 ± 20C and 65±5 R.H). The previous design was replicated three times. At the end of each storage period, the jars containing the flour were sieved thoroughly by 40 wire mesh size to separate the insects. The insects have been counted on the other hand wheat flour was prepared to determine MBQ, EBQ and AFs by HPLC methods. Results: The results indicated the levels of EBQ higher than MBQ in all infested samples at all insect densities (No. of insect pairs). The concentrations of MBQ in wheat flour released by ten adult pairs (10P) with the three storage periods two, three and four months were 10.42 ± 0.56, 22.38 ± 3.67, 27.06 ± 6.71µg/g, respectively. These results increased with insect densities to (30p) were 39.67 ± 0.10, 63.58 ± 2.35 and 106.24 ± 7.4 µg/g after storage periods two, three and four months, respectively. In addition to the concentrations of EBQ with (10P) were 67.45 ± 3.64, 98.0 ± 6.1 204.66 ± 5.8 µg/g with storage periods two, three and four months, respectively. In case (30P) the levels of EBQ were 376.7 ± 0.87, 570.1 ± 2.11 and 1558.66 ± 10.88 (µg/g). The highest concentration of the BQs 1664.90 ± 11.43 (µg/g) released by T. castaneum achieved with the highest adult emergence (1021 insect adult) and the highest insect density (30p) at four months storage period. In general, AFs levels enhanced with a period of storage and insect densities. Conclusion: Levels of the BQs (MBQ and EBQ) increased with an increase of storage periods and insect densities. Therefore, the presence of this insect should be prevented in stored wheat flour reducing AFs contamination is possible by storage for short time and prohibit insects which causes an increase temperature of the flour and moisture, all of which promote production of AFs.

Fate of mycotoxins and related fungi in the anaerobic digestion process

In this study, the possibility to manage maize contaminated with aflatoxins and fumonisins for the production of biogas was considered. This is a priority in the climate change scenario that is expected to increase the occurrence of aflatoxins in maize. The results clearly underline how the anaerobic digestion process used in biogas plants is able to reduce aflatoxin contamination, mainly when highly contaminated maize is used for feeding the biodigestors without affecting the efficiency of methane production. In particular, the higher aflatoxin contamination is, the higher is mycotoxin reduction during biodigestion, with reductions up to 95% in digestate. The co-occurring mycotoxins, fumonisins, were also reduced by around 15%. The vitality of mycotoxin producing fungi was also significantly reduced. Biogas production is therefore suggested as a good alternative use for uncompliant maize.

Effective chemical management for prevention of aflatoxins in maize

The presence of aflatoxins in maize grain has been an increasing problem in the Mediterranean area, possibly due to climate change such as increased temperatures and extended drought periods. It is therefore important to prevent the growth of aflatoxigenic Aspergillus species in the field. There are no fungicides registered for control A. flavus in maize, so this study investigated the efficacy of azoxystrobin, boscalid, cyprodinil, fludioxonil and cyprodinil + fludioxonil to reduce A. flavus growth, sporulation and aflatoxin production in in vitro , and in maize field studies. Based on in vitro inhibition of mycelial growth, the most effective fungicides were cyprodinil (EC 50 0.5 μg mL -1 . Further evaluation of the fungicides on maize seeds infected with A. flavus demonstrated that all the fungicides reduced conidium production by 76 to 94%, and reduced aflatoxin contamination. In a 2-year field study, application of cyprodinil + fludioxonil reduced A. flavus ear rot severity by 40%, and was the most effective formulation for reducing aflatoxin contamination, by 83%. The other four single ingredient fungicides also decreased aflatoxin production on maize kernels (fludioxonil by 80%, cyprodinil by 75%, boscalid by 74% and azoxystrobin by 67%). Field data from this study provide farmers with a new effective chemical approach to control A. flavus and aflatoxin production in maize within an integrated strategy for management of aflatoxins in maize.

Genome sequence and comparative analyses of atoxigenic Aspergillus flavus WRRL 1519

ABSTRACTAflatoxins are toxic secondary metabolites produced by Aspergillus flavus and a few other closely related species of Aspergillus. These highly toxigenic and carcinogenic mycotoxins contaminate global food and feed supplies, posing widespread health risks to humans and domestic animals. Field application of nonaflatoxigenic strains of A. flavus to compete against aflatoxigenic strains has emerged as one of the best management practices for reducing aflatoxins contamination, yielding successful commercial products for corn, cotton seed, and peanuts. In this study, we sequenced the genome and transcriptome of atoxigenic (does not produce aflatoxin or cyclopiazonic acid) A. flavus strain WRRL 1519 isolated from a tree nut orchard to define the genetic characteristics of the strain in relation to aflatoxigenic and other nonaflatoxigenic A. flavus strains. WRRL 1519 strain was similar to other strains in size (38.0 Mb), GC content (47.2%), number of predicted secondary metabolite gene clusters (46), and number of putative proteins (12 121). About 87.4% of the predicted proteome had high shared identity with protein sequences derived from other A. flavus genomes. However, the atoxigenic A. flavus strain WRRL 1519 had deletions, or low shared identity, for many genes in the clusters required for aflatoxins and cyclopiazonic acid (CPA) synthesis. Over half of the aflatoxin synthesis gene cluster was missing, and none of the components of the CPA gene cluster were identified with high sequence similarity. Importantly, the strain appeared to maintain functional sequences of several genes thought to be required for high infectivity. Since the ability to grow on target crop is an important attribute for a successful biocontrol agent, these results indicate that the nonaflatoxigenic A. flavus strain WRRL 1519 would be a good candidate as a biocontrol agent for reducing aflatoxin and CPA accumulation in high-value nut crops.