Aflatoxins are highly carcinogenic secondary metabolites that can contaminate approximately 25% of crops and that cause or exacerbate multiple adverse health conditions, especially in Sub-Saharan Africa and South and Southeast Asia. Regulation and decontamination of aflatoxins in high exposure areas is lacking. Biological detoxification methods are promising because they are assumed to be cheaper and more environmentally friendly compared to chemical alternatives. White-rot fungi produce non-specific enzymes that are known to degrade aflatoxin in in situ and ex situ experiments. The aims of this study were to (1) decontaminate aflatoxin-B-1-(AFB(1)) in naturally contaminated maize with the edible, white-rot fungus Pleurotus ostreatus (oyster mushroom) using a solid-state fermentation system that followed standard cultivation techniques, and to (2) and to assess the risk of mutagenicity in the resulting breakdown products and mushrooms. Vegetative growth and yield characteristics of P. ostreatus were not inhibited by the presence of-AFB(1).-AFB(1) was degraded by up to 94% by the Blue strain. No aflatoxin could be detected in P. ostreatus mushrooms produced from-AFB(1)-contaminated maize. Moreover, the mutagenicity of breakdown products from the maize substrate, and reversion of breakdown products to the parent compound, were minimal. These results suggest that P. ostreatus significantly degrades-AFB(1) in naturally contaminated maize under standard cultivation techniques to levels that are acceptable for some livestock fodder, and that using P. ostreatus to bioconvert crops into mushrooms can reduce-AFB(1)-related losses.

Pre-harvest contamination of maize grain with aflatoxin is a chronic problem worldwide and particularly in the southeastern U.S. Aflatoxin is a mycotoxin produced by the fungus Aspergillus flavus, an opportunistic ear-rot pathogen of maize (Zea mays). Resistance to aflatoxin accumulation is heritable, and resistant germplasm-lines are available. These lines are derived from “exotic” genetic backgrounds and were released as sources of resistance, not parental inbreds. However, all current sources of resistance are quantitative, which complicates conventional efforts to introgress resistance alleles from unadapted but resistant donor lines to adapted but susceptible recipient lines. Mapping quantitative trait loci (QTL) and their linked markers enables targeted introgression of the desired alleles via marker-assisted selection. Quantitative trait loci were identified in two F2:3 mapping populations, derived from crossing resistant inbreds Mp715 and Mp717 to a common susceptible parent (Va35). The Mp715 x Va35 population was phenotyped for aflatoxin accumulation under artificial inoculation in replicated field trials at Mississippi State (MSU) in 2015 and 2016. The Mp717 x Va35 population was phenotyped at MSU and Lubbock, TX in 2016. Populations were genotyped using simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers and linkage maps created in JoinMap4. To locate QTL, linkage maps, genotypes, and phenotypes were analyzed jointly in QTL Cartographer 2.5 using composite interval mapping (CIM) and multiple interval mapping (MIM) procedures. Five QTL with the beneficial allele contributed by Mp715 were identified during CIM in bins 5.01, 6.06, 7.03 10.04 and 10.05. Three QTL with the beneficial allele contributed by Mp717 were identified during CIM in bins 3.07/3.08, 7.02/7.03, and 10.05. In both populations, QTL were identified with the beneficial allele contributed by Va35. Those QTL did not co-locate across populations but four of the six were on chromosome 1. Significant QTL effects from CIM were used as the initial model terms in MIM, where all QTL effects were fit simultaneously and their gene-action and epistatic interactions estimated.

Maize (Zea mays L.) is one of the world’s largest food crops and thus any pathogens of maize are of great importance. Aspergillus flavus is one of these pathogens and it produces a carcinogenic metabolite called aflatoxin. Efforts to reduce infection by A. flavus and subsequent aflatoxin accumulation include the development of maize lines resistant to aflatoxin accumulation. However, resistant lines that have been developed contain agronomically unfavorable traits. Gene-based markers would allow for easier transfer of resistance from resistant inbred lines into maize lines with good agronomic traits. The focus of this research was the development of gene-based markers for resistance to aflatoxin accumulation. To this end, two genes were characterized for their association with reduced aflatoxin accumulation in maize. A gene coding for a photosytem II3 protein shown to be differentially regulated between maize lines Mp313E (resistant) and Va35 (susceptible) was used to develop the marker MpM1. This marker was shown to be associated with resistance to aflatoxin accumulation in three F2:3 mapping populations derived from Mp313E x B73, Mp313E x Va35, and Mp715 x T173 and identified a new quantitative trait locus (QTL) on chromosome 4. The second gene chosen was the chitinase A gene (chiA), which has been shown to inhibit fungal growth and is differentially regulated between resistant and susceptible lines of maize. ChiA also had an association with reduced aflatoxin accumulation in the three F2:3 mapping populations and identified a new QTL in the Mp313E x Va35 population. Together, MpM1 and chiA were associated with 27% of the phenotypic variation in one environment of the Mp313E x B73 population. These markers represent the first two gene-based markers developed for resistance to aflatoxin accumulation, and the methodology developed in this study can be used to screen other candidate genes for potential use as gene-based makers.

In international trade involving agricultural products, attempts to safeguard the health of humans, animals, and plants, have led to the imposition of sanitary and phytosanitary (SPS) standards. Due to the fact that groundnuts are susceptible to aflatoxin contamination, stringent aflatoxin standards have been imposed on groundnut trade by many developed countries. For Senegal and other groundnut exporters in the developing world, these aflatoxin standards pose a major challenge. As a result, in Senegal's confectionery groundnut sector, CIRAD (a French scientific organization) has commenced research aimed at developing an aflatoxin-reducing program. This study evaluates the potential economic impact of CIRAD's aflatoxin-reducing program. The hypotheses underlying the study are as follows: (i) The adoption of CIRAD's aflatoxin-reducing program would enhance the welfare of Senegal's confectionery groundnut farmers (ii) An overall welfare net-gain would be derived by Senegal from the adoption of the program. The analysis employs an economic surplus model that incorporates trade, as well as, domestic production and consumption. Various scenarios of program-effectiveness are examined. The results support the hypotheses of the study; besides enhancing farmers' welfare, the adoption of the aflatoxin-reducing program is expected to yield an overall net-gain ranging between US$0.56 million and US$4.25 million. The overall net-gain is, however, very small. / Master of Science

Aflatoxins, toxic secondary metabolites produced by some Aspergillus species, are a universal agricultural economic problem and a critical health issue. Despite decades of control efforts, aflatoxin contamination is responsible for a global loss of millions of tons of crops each year. We show that host-induced gene silencing is an effective method for eliminating this toxin in transgenic maize. We transformed maize plants with a kernel-specific RNA interference (RNAi) gene cassette targeting the aflC gene, which encodes an enzyme in the Aspergillus aflatoxin biosynthetic pathway. After pathogen infection, aflatoxin could not be detected in kernels from these RNAi transgenic maize plants, while toxin loads reached thousands of parts per billion in nontransgenic control kernels. A comparison of transcripts in developing aflatoxin-free transgenic kernels with those from nontransgenic kernels showed no significant differences between these two groups. These results demonstrate that small interfering RNA molecules can be used to silence aflatoxin biosynthesis in maize, providing an attractive and precise engineering strategy that could also be extended to other crops to improve food security.

Aspergillus flavus is the most common causal agent of aflatoxin contamination of food and feed. However, aflatoxin-producing potential varies widely among A. flavus genotypes with many producing no aflatoxins. Some non-aflatoxigenic genotypes are used as biocontrol agents to prevent contamination. Aflatoxin biosynthesis genes are tightly clustered in a highly conserved order. Gene deletions and presence of single nucleotide polymorphisms (SNPs) in aflatoxin biosynthesis genes are often associated with A. flavus inability to produce aflatoxins. In order to identify mechanisms of non-aflatoxigenicity in non-aflatoxigenic genotypes of value in aflatoxin biocontrol, complete cluster sequences of 35 A. flavus genotypes from Africa and North America were analyzed. Inability of some genotypes to produce aflatoxin resulted from deletion of biosynthesis genes. In other genotypes, non-aflatoxigenicity originated from SNP formation. The process of degeneration differed across the gene cluster; genes involved in early biosynthesis stages were more likely to be deleted while genes involved in later stages displayed high frequencies of SNPs. Comparative analyses of aflatoxin gene clusters provides insight into the diversity of mechanisms of non-aflatoxigenicity in A. flavus genotypes used as biological control agents. The sequences provide resources for both diagnosis of non-aflatoxigenicity and monitoring of biocontrol genotypes during biopesticide manufacture and in the environment.

Foi realizado um experimento no aviário experimental do Departamento de Zootecnia da Universidade de São Paulo (USP), na Faculdade de Zootecnia e Engenharia de Alimentos (FZEA), em Pirassununga/SP com o objetivo de avaliar o efeito de dois níveis vitamínicos (comercial e OVN) com ou sem aflatoxina em dietas de frangos de corte no período de 1 a 42 dias. Foram utilizados 1800 pintinhos, machos, Cobb 500, distribuídos em um delineamento inteiramente casualizado em esquema fatorial 2 x 2 x 2 (2 níveis vitamínicos - comercial e OVN, 2 níveis de aflatoxina - 0 ppm e 0,5 ppm, e 2 níveis de adsorventes - 0 e 10 kg/ton), totalizando 8 tratamentos com 15 repetições de 15 aves cada. As dietas foram fornecidas fareladas e a base de milho e farelo de soja, formuladas segundo os níveis praticados por uma integradora da região. Para avaliar o desempenho foram analisados o consumo de ração, ganho de peso e conversão alimentar de 1 a 49 dias. Para avaliação de carcaça (rendimento de carcaça, peito e pernas), determinação de incidência de BBS e determinação do peso das vísceras abdominais e coração foram abatidas duas aves por repetição aos 45 dias. Os resultados mostraram que frangos de corte, machos, alimentados com nível OVN de vitaminas, apresentaram melhor ganho de peso, conversão alimentar, rendimento de carcaça e peito quando comparado com o nível comercial de vitaminas (P<0,05) e que as dietas contendo 0,5 ppm de aflatoxinas resultaram em menor ganho de peso, rendimento de carcaça, porcentagem de peito e aumentou o tamanho do coração e fígado do animal (P<0,05). O uso de 10kg/ton de adsorvente só apresentou resultado positivo no final da vida dos animais (dos 39 a 49 dias) (P<0,05) e somente na conversão alimentar. Esse estudo permite concluir que a aflatoxina resulta em perdas de desempenho e rendimento de carcaças e que o fornecimento de níveis ótimos de vitaminas melhora os resultados dessas características. O uso de adsorventes se mostrou inviável nesse estudo. / An experiment was conducted in an experimental aviary the Department of Animal Science, University of São Paulo (USP), the Faculty of Animal Science and Food Engineering (FZEA) in Pirassununga/SP, to evaluate the performance, carcass characteristics and weight of offal in broiler chickens fed with two levels of vitamins (commercial and VNO) with or without aflatoxin in broiler diets. Were used 1800 chicks, male, Cobb 500 distributed in a completely randomized 2 x 2 x 2 factorial arrangement (two vitamin levels, two levels of aflatoxin and two levels of adsorbents), totaling eight treatments with 15 replicates of 15 birds each. Diets were fed mash and corn and soybean meal, formulated according to the levels charged by an integrator in the region. To evaluate the performance were analyzed feed intake, weight gain, feed conversion from 1 to 49 days. For evaluation of carcass yield (carcass, breast and legs), determination of the incidence of BBS and determination of the weight of the abdominal viscera and heart were killed two birds per replicate at 45 days. The results showed that broilers, males fed VNO level of vitamins showed better weight gain, feed conversion, carcass yield and breast when compared to the commercial level of vitamins (P<0.05) and that diets intoxicated with 0.5 ppm of aflatoxin resulted in less weight gain, carcass yield, percentage of breast and increased the size of the heart and liver of the animal (P<0.05). The use of adsorbent 10kg/ton only had a positive result at the end of life of animals (from 39 to 49 days) (P<0.05) and only in the feed. This study indicates that aflatoxin results in loss of performance and carcass yield and the provision of optimal levels of vitamins improved the results of these characteristics. The use of adsorbents in this study proved to be unfeasible.

ArtinM é uma lectina ligante a carboidrato D-manose que se interage a receptores de células fagocíticas induzindo a produção de mediadores pró- inflamatórios relacionados à resposta imune antitumoral. Aflatoxinas são micotoxinas produzidas por fungos do gênero Aspergillus. A Aflatoxina B1 (AFB1) é a toxina sintetizada mais abundantemente e a que apresenta o maior poder toxigênico, sendo capaz de induzir carcinoma hepatocelular (CHC) em humanos. O objetivo deste estudo foi investigar o papel da lectina ArtinM na hepatocarcinogênese induzida pela AFB1 em ratos. Setenta e dois ratos recém-desmamados foram divididos em três grupos: Controle - animais tratados com veículo; AFB1 - animais intoxicados com AFB1; AFB1+ArtinM - animais tratados com AFB1 e ArtinM. Ratos Wistar foram intoxicados por gavagem com 400 ?g de AFB1 por quilograma de ração ingerida durante três meses, enquanto o grupo AFB1+ArtinM recebeu adicionalmente três doses da lectina por via subcutânea (50 ?g por quilograma de peso do animal por dose) nos 45, 60 e 75 após inicio do experimento. Animais foram eutanasiados 3 e 12 meses após início das gavagens. A expressão hepática de proteínas relacionadas à hepatocarcinogênese foi avaliada por técnicas de imunohistoquímica, Western blotting e PCR em tempo real nos animais eutanasiados após 3 meses de intoxicação. A incidência de lesões pré-neoplásicas e de tumores hepáticos foi mensurada 3 e 12 meses após início das gavagens, respectivamente. Os animais tratados com ArtinM apresentaram maior expressão hepática de proteínas supressoras tumorais além de redução do número de focos pré-neoplásicos e de tumores hepáticos em relação aos animais que receberam apenas a micotoxina. Conclui-se, portanto, que ArtinM possui efeito protetor durante o processo de hepatocarcinogênese induzida por AFB1. / ArtinM is a D-mannose carbohydrate-binding lectin that interacts with phagocytic cell receptors inducing the production of pro-inflammatory mediators related to the antitumor immune response. Aflatoxins are mycotoxins produced by fungi of the genus Aspergillus. Aflatoxin B1 (AFB1) is the toxin most abundantly synthesized and the one with the highest toxigenic power, being able to induce hepatocellular carcinoma (HCC) in humans. The aim of this study was to investigate the role of ArtinM lectin in AFB1-induced hepatocarcinogenesis in rats. Seventy-two newly weaned rats were divided into three groups: Control - vehicle-treated animals; AFB1 - animals poisoned with AFB1; AFB1 + ArtinM - animals treated with AFB1 and ArtinM. Wistar rats were gavage-poisoned with 400 ?g AFB1 per kilogram of ration fed for three months, while the AFB1 + ArtinM group received three subcutaneous doses of the lectin (50 ?g per kilogram of animal weight per dose) 45, 60 and 75 days after the start of the experiment. Animals were euthanized 3 and 12 months after initiation of treatments. Hepatic expression of hepatocarcinogenesis-related proteins was assessed by immunohistochemistry, Western blotting, and realtime PCR in euthanized animals after three months of intoxication. The incidence of pre-neoplastic lesions and liver tumors was measured 3 and 12 months after the start of treatments, respectively. Animals treated with ArtinM had fewer pre-neoplastic foci and hepatic tumors than the animals that receiving mycotoxin alone, as well as showing greater hepatic expression of tumor suppressor proteins. It is concluded, therefore, that ArtinM has a protective effect during the process of hepatocarcinogenesis induced by AFB1.

ArtinM é uma lectina ligante a carboidrato D-manose que se interage a receptores de células fagocíticas induzindo a produção de mediadores pró- inflamatórios relacionados à resposta imune antitumoral. Aflatoxinas são micotoxinas produzidas por fungos do gênero Aspergillus. A Aflatoxina B1 (AFB1) é a toxina sintetizada mais abundantemente e a que apresenta o maior poder toxigênico, sendo capaz de induzir carcinoma hepatocelular (CHC) em humanos. O objetivo deste estudo foi investigar o papel da lectina ArtinM na hepatocarcinogênese induzida pela AFB1 em ratos. Setenta e dois ratos recém-desmamados foram divididos em três grupos: Controle - animais tratados com veículo; AFB1 - animais intoxicados com AFB1; AFB1+ArtinM - animais tratados com AFB1 e ArtinM. Ratos Wistar foram intoxicados por gavagem com 400 ?g de AFB1 por quilograma de ração ingerida durante três meses, enquanto o grupo AFB1+ArtinM recebeu adicionalmente três doses da lectina por via subcutânea (50 ?g por quilograma de peso do animal por dose) nos 45, 60 e 75 após inicio do experimento. Animais foram eutanasiados 3 e 12 meses após início das gavagens. A expressão hepática de proteínas relacionadas à hepatocarcinogênese foi avaliada por técnicas de imunohistoquímica, Western blotting e PCR em tempo real nos animais eutanasiados após 3 meses de intoxicação. A incidência de lesões pré-neoplásicas e de tumores hepáticos foi mensurada 3 e 12 meses após início das gavagens, respectivamente. Os animais tratados com ArtinM apresentaram maior expressão hepática de proteínas supressoras tumorais além de redução do número de focos pré-neoplásicos e de tumores hepáticos em relação aos animais que receberam apenas a micotoxina. Conclui-se, portanto, que ArtinM possui efeito protetor durante o processo de hepatocarcinogênese induzida por AFB1. / ArtinM is a D-mannose carbohydrate-binding lectin that interacts with phagocytic cell receptors inducing the production of pro-inflammatory mediators related to the antitumor immune response. Aflatoxins are mycotoxins produced by fungi of the genus Aspergillus. Aflatoxin B1 (AFB1) is the toxin most abundantly synthesized and the one with the highest toxigenic power, being able to induce hepatocellular carcinoma (HCC) in humans. The aim of this study was to investigate the role of ArtinM lectin in AFB1-induced hepatocarcinogenesis in rats. Seventy-two newly weaned rats were divided into three groups: Control - vehicle-treated animals; AFB1 - animals poisoned with AFB1; AFB1 + ArtinM - animals treated with AFB1 and ArtinM. Wistar rats were gavage-poisoned with 400 ?g AFB1 per kilogram of ration fed for three months, while the AFB1 + ArtinM group received three subcutaneous doses of the lectin (50 ?g per kilogram of animal weight per dose) 45, 60 and 75 days after the start of the experiment. Animals were euthanized 3 and 12 months after initiation of treatments. Hepatic expression of hepatocarcinogenesis-related proteins was assessed by immunohistochemistry, Western blotting, and realtime PCR in euthanized animals after three months of intoxication. The incidence of pre-neoplastic lesions and liver tumors was measured 3 and 12 months after the start of treatments, respectively. Animals treated with ArtinM had fewer pre-neoplastic foci and hepatic tumors than the animals that receiving mycotoxin alone, as well as showing greater hepatic expression of tumor suppressor proteins. It is concluded, therefore, that ArtinM has a protective effect during the process of hepatocarcinogenesis induced by AFB1.

Aflatoxin is a potent biological toxin produced by fungi Aspergillus flavus and A. parasiticus. Current quantification methods for aflatoxins are mostly established on immunoaffinity columns which are both costly and labor intensive. Inspired by smectites’ high aflatoxin adsorption capacity and affinity, a novel aflatoxin quantification sensor based on smectite-polyacrylamide (PAM) nanocomposite was fabricated. First, a smectite-PAM nanocomposite film was synthesized on flat silicon substrates which assembled smectite particles from the clay suspension. A layer-by-layer assembly process was developed to achieve uniform morphology and thickness of the nanocomposite films. During the aflatoxin quantification process, positive correlations between the fluorescence intensity from the aflatoxin B1 (AFB1) adsorbed smectite-PAM nanocomposite films and the AFB1 concentration in the test solutions were obtained. The smectite-PAM nanocomposite film has shown similar AFB1 adsorption capabilities as the smectite. Second, the smectite-PAM nanocomposite film was optimized in order to achieve the aflatoxin quantification at ppb level (below 20ppb) in corn extraction solutions. The smectite was modified by Ba2+, which had demonstrated to be able to improve its aflatoxin adsorption capacity. PAM aqueous solutions with the mass concentration ranging from 0.8% to 0.001% were tested. The results showed that the nanocomposite synthesized from 0.005% concentration of PAM solution generated the best properties. After the optimization, the smectite-PAM nanocomposite films achieved the detection of aflatoxin B1, B2, G1 and G2 (AFB2, AFG1 and AFG2) in 10 ppb corn extraction solution. Aflatoxin quantifications in AFB1 and AFB2 mixture solution, AFB1 and AFB2 mixture solution and AFB1 and AFG1 mixture solution were conducted, and the recoveries of last test ranged from 90.52% to 110.11% at low aflatoxin concentration (below 20 ppb). Third, in order to shorten the quantification duration and simplify the detection process, a novel aflatoxin detection array based on smectite-PAM nanocomposite and an improved fluorometric quantification method were developed. Through a microfluidic chip, the reaction time was reduced to 10~20min. Two concentration levels (20~80ppb/5~15ppb) of aflatoxin B1 spiked corn extraction solutions were tested. In the fluorometric quantification step, a common lab-use 365 nm ultraviolet lamp replaced the spectrofluorometer which simplified and accelerated the process.